Tissue engineering of lung

ABSTRACT

The present invention relates to compositions comprising a decellularized tissue. The present invention also provides an engineered three dimensional lung tissue exhibiting characteristics of a natural lung tissue. The engineered tissue is useful for the study of lung developmental biology and pathology as well as drug discovery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/625,080, filed Feb. 18, 2015, now U.S. Pat. No. 10,188,683, which iscontinuation of U.S. patent application Ser. No. 13/146,605, filed Oct.10, 2011, now abandoned, which is a U.S. national phase applicationfiled under 35 U.S.C. § 371 claiming benefit to International PatentApplication No. PCT/US2010/023213, filed on Feb. 4, 2010, which isentitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication No. 61/206,799, filed on Feb. 4, 2009, each of whichapplication is hereby incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Every year, 400,000 Americans die of lung disease. Of further concern,the death rate due to lung disease is increasing, while the death ratesfor the other major disease categories are decreasing (heart disease,cancer and stroke). For several lung diseases, including cysticfibrosis, emphysema/COPD, and idiopathic pulmonary fibrosis, lungtransplantation remains the only definitive treatment. However, patientsurvival after lung transplant is only 50% at 5 years and 24% at 10years [Mondrinos et al., 2008, Tissue Eng 14:361-8]. There is thereforegreat demand for the development of engineered lung tissue that could beused for transplantation. One advantage of engineered lung tissue isthat the tissue can be grown using a patient's own cells, therebyavoiding the need for strong immunosuppression, as is required withcurrent lung transplantation. Immunosuppression is necessary to preventrejection of the transplanted organ, but can lead to a wide range ofproblems, including infection, malignancy, kidney impairment,cardiovascular problems, and neurologic disorders [Pietra et al., 2000,J Clin Invest 106:1003-10; Christie et al., 2009, J Heart LungTransplant 28:1031-49].

Tissue engineering is a growing field that seeks to combine cellular,molecular, technological and medical advances to create replacementtissues suitable for implantation. Promising work has been done on avariety of tissues, including blood vessels, urinary bladder, heartvalves, and cardiac tissue [Nichols et al, 2008, Proc Am Thor Soc5:723-30; Satchell et al., 2004, J Am Soc Nephrol 15:566-74; Atala etal., 2006, Lancet 367:1241-6; Orfanos et al., 2004, Intensive Care Med30:1702-14]. However, lung is a difficult tissue to engineer in thelaboratory. Lung requires a complex matrix that can withstand themechanical pressures of breathing, that can support the growth ofendothelial, epithelial and mesenchymal cells, and that provides a meansfor gas exchange between two very different yet intimately juxtaposedcompartments.

Besides potential patient use in clinical settings, engineered lungtissue can be used in the laboratory to study a wide variety ofimportant aspects of pulmonary biology and physiology. There are veryfew in vitro 3-dimensional lung culture models [Vandenbroucke et al.,2008, Ann N Y Acad Sci 1123:134-45]. Furthermore, pulmonary endothelialand epithelial cells are more difficult to culture in the laboratorythan many other cell types [Malda et al., 2004, Biomaterials 25:5773-80;Reichenspurner, 2005, J Heart Lung Transplant 24:119-30], and there hasbeen relatively slow progress in the field of pulmonary progenitor andstem cell biology [Blaisdell et al., 2009, Stem Cells 27:2263-70;Muratore et al., 2008, J Surg Res 155(2):225-30]. Thus, there is a needin the art for the development of an in vitro lung tissue thatreplicates key features of the native pulmonary environment. The presentinvention satisfies this need in the art.

BRIEF SUMMARY OF THE INVENTION

The invention provides a decellularized tissue capable of supportingcell growth. Preferably, the decellularized tissue exhibits acharacteristic of a corresponding natural tissue prior todecellularization. More preferably, the tissue is a lung.

In one embodiment, the decellularized tissue exhibits a morphologysubstantially similar to that of an otherwise identical tissue prior todecellularization.

In another embodiment, the decellularized tissue of claim 1 retaining anextracellular matrix of said corresponding natural tissue, wherein saidextracellular matrix comprises an exterior surface, and wherein saidexterior surface is substantially intact.

In another embodiment, immunogenic markers have been substantiallyremoved from the decellularized tissue.

In one embodiment, the decellularized tissue exhibits mechanicalproperties substantially similar to that of a corresponding naturaltissue.

The invention provides a composition comprising a three dimensionalscaffold and a population of cells. Preferably, the composition iscapable of supporting and maintaining the differentiation state of alung cell.

In one embodiment, the three dimensional scaffold is a decellularizedtissue.

In another embodiment, the composition exhibits an intact airway treeand vascular network.

In another embodiment, the population of cells comprises a stem cell.

In another embodiment, the population of cells comprises epithelial andendothelial cells.

In another embodiment, the cells are genetically modified. In oneembodiment, the cell is genetically modified to express the CFTR gene.

In one embodiment, the composition is capable of supporting andmaintaining the differentiation state of an alveolar epithelial cell.

In another embodiment, the scaffold comprises a biocompatiable materialselected from the group consisting of fibronectin, laminin, collagen,glycoprotein, thrombospondin, elastin, fibrillin, mucopolysaccharide,glycolipid, heparin sulfate, chondroitin sulfate, keratin sulfate,glycosaminoglycan, hyaluronic acid, proteoglycan, vitronectin,poly-D-lysine, polysaccharide, and combinations thereof.

In one embodiment, the cells exhibit gene expression associated withinduction of branching morphogenesis.

In another embodiment, the composition comprises a characteristic of alung tissue. In some instances, the characteristic is selected from thegroup consisting of branching morphogenesis, distal lung epithelialcytodifferentiation, epithelial growth, vascular development, andcombinations thereof.

The invention provides a method of making an engineered threedimensional tissue capable of supporting and maintaining thedifferentiation state of a lung cell. The method comprises seeding adecellularized scaffold with a population of cells to produce a seededscaffold.

The invention provides an in vitro method for screening a test agent forthe ability of the test agent to modulate the health of a lung tissue.The method comprises contacting a test agent to an engineered threedimensional lung tissue model and measuring the effect the test agenthas on the model. Any alteration to the model is an indication that thetest agent is able to modulate the health of a lung tissue.

In one embodiment, the test agent is selected from the group consistingof a chemical agent, a pharmaceutical, a peptide, a nucleic acid, andradiation.

In another embodiment, the test agent is a delivery vehicle for atherapeutic agent.

In one embodiment, the method comprises determining the effect of thetest agent on cell number, area, volume, shape, morphology, markerexpression or chromosomal fragmentation.

In another embodiment, the method comprises the step of selecting anagent which has a desired effect on the lung tissue model.

The invention provides a method of alleviating or treating a lung defectin a mammal. The method comprises administering to a mammal atherapeutically effective amount of a composition comprising a threedimensional construct capable of supporting and maintaining thedifferentiation state of an lung cell, thereby alleviating or treatingthe lung defect in the mammal.

The invention provides an implantable composition comprising adecellularized tissue capable of supporting cell growth. Preferably, thedecellularized tissue exhibits a characteristic of a correspondingnatural tissue prior to decellularization.

In one embodiment, the implantable composition comprises a population ofcells. Preferably, the implantable composition is capable of supportingand maintaining the differentiation state of a lung cell.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIGS. 1A through 1D are a series of images depicting H&E staining andquantitative DNA assay of native and decellularized lung. FIG. 1demonstrates that cellular material was removed yet the architecture ofthe scaffold was retained. DNA was removed to −1.2% of native levels. *indicates p<0.01. FIG. 1D is an image of decellularized lung.

FIGS. 2A and 2B are a series of images depicting staining for remnantDNA in decellularized scaffolds. DNA is stained using DAPI. Images wereexposed for the same time to enable comparison.

FIG. 3 is a Western blot for MHC Class I and II antigen, demonstratinglack of MHC Class I or II antigen in decellularized scaffolds.

FIGS. 4A and 4B are a series of images depicting collagen staining innative and decellularized lung. Collagen I is stained green, collagen IVis stained red, and nuclei are counterstained blue with DAPI. Collagen Iis found near large vessels while collagen IV is distributed throughoutthe parenchyma. Note that in native lung, red blood cells in theparenchyma appear green due to autofluorescence.

FIGS. 5A and 5B are a series of images depicting scanning EM of nativeand decellularized lung. Alveolar septae are intact. Scale bars are 100μm in left panels and 20 μm in right panels.

FIGS. 6A through 6C are a series of images depicting transmission EM ofnative and decellularized lung. The alveolar basement membrane isretained when decellularization perfusion pressure is maintained below30 mmHg. C indicates capillaries, A indicates alveoli, and S indicatesthe alveolar septae. Scale bars are 2 μm in all panels.

FIG. 7 is an image depicting transmission EM of decellularized lungdemonstrating preserved capillaries. Perfusion pressure fordecellularization was less than 20 mmHg. C indicates capillaries, whileA indicates alveoli. Dimensions of alveoli and capillaries appearsmaller than occur in vivo, due to compression of the decellularizedmatrix. Scale bars are 2 μm on top panels, 1 μm on bottom left panel,and 500 nm on bottom right panel.

FIG. 8 is a graph depicting retention of 5 μm microspheres bydecellularized scaffolds. Microsphere assay demonstrates that lowperfusion pressure (<30 mmHg) during decellularization enables theretention of 95% of 5 μm particles in the airway compartment. *indicates p<0.05 compared to native.

FIGS. 9A and 9B are a series of images of a Micro CT of the vasculatureof native and decellularized lung. Overall, decellularized scaffoldsappear similar to native, when imaged with a resolution of 58 μm.

FIGS. 10A and 10B are a series of images depicting high resolution microCT of the vasculature of native and decellularized lung. Resolution ofthese scans is 6.5 μm.

FIG. 11 is a schematic depicting a mechanical testing protocol. Briefly,a strip of lung tissue is attached to the upper plate, which is thenlowered and the tissue attached to the lower plate. The tissue iscyclically stretched to 20% strain and then stretched until failure.

FIGS. 12A through 12C are a series of images depicting collagen stainingand content of native and decellularized lung. Masson's trichrome stainreveals wavy dark blue fibers in both native and decellularized lung.Quantitative assay demonstrates preservation of collagen in native anddecellularized lungs, but loss of collagen after decellularization usingsodium dodecylsulfate (SDS). * indicates p<0.01.

FIGS. 13A through 13C are a series of images depicting elastinhistochemistry (Verhoff-van Geison) for native and decellularized lung.FIG. 13 depicts wavy dark elastin fibers in both native anddecellularized lung. Quantitative assay demonstrates preservation ofsome elastin in decellularized lungs compared to native. * indicatesp<0.01.

FIGS. 14A through 14C are a series of images depicting GAGhistochemistry (Alcian blue) for native and decellularized lung.Depicted is blue GAG staining in native lung but their absence indecellularized lung. Quantitative assay demonstrates loss of sulfatedGAGs in decellularized lungs compared to native lung. * indicatesp<0.01.

FIGS. 15A and 15B are an image depicting stress-strain curves of nativeand decellularized lung. SDS indicates a lung treated with sodiumdodecylsulfate.

FIG. 16 is a chart depicting ultimate tensile strengths of native,decellularized and SDS-decellularized lung. SDS indicates a lungdecellularized using sodium dodecylsulfate. * indicates p<0.01 comparedto native.

FIGS. 17A and 17B are a series of images depicting schematic diagrams ofthe bioreactor used for in vitro lung culture.

FIGS. 18A and 18B are a series of images depicting pulmonary artery andtracheal pressures during in vitro lung culture. Perfusion rate is ˜5ml/min.

FIGS. 19A through 19C are a series of images depicting the effect ofventilation with air versus liquid on lung architecture and airwayepithelium. Air ventilation causes airway dilation and destruction ofthe airway epithelium after a 3 day culture.

FIGS. 20A and 20B are a series of images depicting the effect ofvascular perfusion and pressure on cell apoptosis and cell number duringnative lung culture. * indicates p<0.01 and # indicates p<0.05 comparedto native.

FIGS. 21A through 21D are a series of images depicting a comparison ofCCSP and SPC expression in native lung and perfused cultured lung. CCSPand SPC are stained in red, with nuclei counterstained blue with DAPI.

FIGS. 22A and 22B are a series of images depicting a comparison of PECAMexpression in native lung and perfused cultured lung. PECAM expressionis still noted for perfused lung culture (30 mmHg). PECAM is stainedred, with nuclei counterstained blue with DAPI.

FIGS. 23A and 23B are a series of images depicting the effect ofventilation on cell apoptosis and cell number during native lungculture. * indicates p<0.01 and # indicates p<0.05 compared to native.

FIGS. 24A through 24C are a series of images depicting apoptotic nucleiin native and ventilated cultured lung. Ventilation with a singleconnection led to a much higher rate of apoptotic nuclei, as compared tonative lung or ventilation with an airway ‘loop’. Apoptotic nuclei arestained brown via TUNEL, with normal nuclei counterstained green.

FIGS. 25A through 25J are a series of images depicting alveolarstructure in native and 7-day cultured lung. Cell morphology, alveolarstructure, and septal architecture appear similar between native andcultured, ventilated lung. FIGS. 25C through 25J depicts maintenance ofpulmonary cell differentiation after 7 days of in vitro ventilated lungculture.

FIG. 26 is an image demonstrating that ventilation enables passiveperfusion of pulmonary vasculature. Microspheres are found in vesselsand capillaries due solely to ventilatory motions of the lung during invitro culture.

FIGS. 27A and 27B are a series of images depicting H&E stain of theimmortalized epithelial cell line MLE-12 cultured on decellularizedscaffolds.

FIGS. 28A through 28F are a series of images depicting flow cytometrystaining of a panel of pulmonary markers of isolated neonatal pulmonarycells. Blue or green curves are isotype control stains and red is theantigen indicated.

FIG. 29 is an image depicting H&E stain of engineered lung at 8 days ofculture. Conditions here are optimized for epithelial cell growth.

FIGS. 30A and 30B are a series of images depicting PCNA staining ofengineered lung at 4 and 8 days of culture. Proliferating nuclei stainbrown for PCNA; negative nuclei are counterstained with hematoxylin.

FIGS. 31A and 31B are a series of images depicting TUNEL staining ofengineered lung at 4 and 8 days of culture. Positive nuclei are brown,while negative nuclei are counterstained with methyl green.

FIGS. 32A through 32C are a series of images depicting Clara Cellsecretory protein (CCSP) staining of native and engineered lung at 4days. CCSP is stained red, while nuclei are counterstained blue withDAPI.

FIGS. 33A through 33C are a series of images depicting surfactantprotein C staining of native and engineered lung at 4 days and 8 days.SPC is stained red, while nuclei are counterstained blue with DAPI.

FIGS. 34A through 34C are a series of images depicting aquaporin-5staining of native and engineered lung at 4 days. AQP is stained red,while nuclei are counterstained blue with DAPI.

FIGS. 35A and 35B are a series of images depicting dual staining for SPCand CCSP in engineered lung tissue. SPC is stained green, CCSP isstained red, and nuclei are counterstained blue with DAPI. SPC-CCSP dualpositive cells appear yellow.

FIGS. 36A through 36C are a series of images depicting cytokeratin-14staining for basal cells in native and engineered lung. Cytokeratinstains red, while nuclei are counterstained blue with DAPI.

FIG. 37 is an image depicting dual staining for cytokeratin-14 and CCSPin engineered lung. Cytokeratin-14 is stained red, CCSP is stainedgreen, and nuclei are counterstained blue with DAPI.

FIGS. 38A and 38B are a series of images depicting α-actin staining ofnative and engineered lung. α-actin is stained green, while nuclei arecounterstained blue with DAPI.

FIGS. 39A through 39F are a series of images depicting the effect ofmedia composition on epithelial development. Epithelial structures aredriven towards apical expression of SPC granules with loss of CCSPexpression when cultured in BGJb media. In DMEM media, cells retainexpression of both SPC and CCSP, with SPC expression diffuselycytoplasmic.

FIG. 40 is an image depicting surfactant expression in engineeredepithelial tissues. ‘Lad’ is a protein ladder; the indicated bands are20 and 25 kDa; ‘Nat’ is native lung tissue; ‘Vent’ is engineered lungtissue ventilated with DMEM medium; ‘Perf’ is engineered lung tissueperfused with DMEM medium; ‘DMEM’ is statically cultured engineered lungin DMEM medium; ‘BGJb’ is statically cultured engineered lung in BGJbmedium; ‘ALI’ are engineered lung ventilated with air; and ‘Decell’ isdecellularized scaffold.

FIGS. 41A through 41C are a series of images depicting the effect ofventilation with air on epithelial development in engineered lungtissue. AQP expression is noted in parenchymal cells (top left) that arealso positive for SPC (FIG. 41B), as well as occasional strongexpression in cuboidal epithelial cells (top right). CCSP expression ofcuboidal epithelium is also noted (FIG. 41C).

FIGS. 42A and 42B are a series of images depicting ciliated epitheliumin native and engineered lung. Ciliated cells are highlighted in red forengineered lung.

FIGS. 43A and 43B are a series of images depicting the effect ofperfusion and ventilation on engineered lung culture.

FIGS. 44A through 44D are a series of images depicting the effect ofperfusion and ventilation on cell proliferation and apoptosis inengineered lung culture.

FIGS. 45A and 45B are a series of images depicting the effect ofperfusion and ventilation on CCSP expression in engineered lung tissue.CCSP is stained red, while nuclei are counterstained blue with DAPI.

FIGS. 46A through 46B are a series of images depicting the effect ofperfusion and ventilation on SPC expression in engineered lung tissue.SPC is stained in red, and nuclei are counterstained blue with DAPI.

FIG. 47 is an image depicting an H&E stain of a fibronectin-coateddecellularized scaffold seeded with rat lung microvascular endothelialcells.

FIGS. 48A and 48B are a series of images depicting H&E staining ofperfused versus ventilated engineered lung endothelium.

FIGS. 49A and 49B are a series of images depicting TUNEL staining ofperfused versus ventilated engineered lung endothelium. EC cultured withventilation only are substantially more apoptotic than perfused lung.Apoptotic nuclei stain brown via TUNEL while negative nuclei arecounterstained with methyl green.

FIG. 50 is an image demonstration of tight junction formation betweenendothelial cells in engineered lung tissue. Endothelial cells aremarked with asterisks, separated by an extended cell-cell junction.Scale bar is 500 nm.

FIGS. 51A and 51B are a series of images depicting expression ofVE-cadherin in native and engineered lung. VE-cadherin is stained red,with nuclei counterstained blue with DAPI.

FIG. 52 is a chart depicting the permeability of engineered lungs seededwith endothelial cells alone to 2 megadalton FITC-labelled dextrans. *indicates p<0.05 compared to decellularized scaffolds.

FIG. 53 is a chart depicting the ultimate tensile strength of engineeredtissues. Native and decellularized lung strengths are also shown.

FIGS. 54A through 54C are a series of images depicting medium impactsthe growth of engineered endothelial tissue. Engineered perfusedendothelium was cultured in the indicated medium type. H&E histology isshown in the left panels, while right panels show apoptotic nuclei inbrown (via TUNEL) while normal nuclei are counterstained with methylgreen.

FIG. 55 is a series of images demonstrating that decellularized tracheaprepared with incubation in CHAPS buffer for 4-8 hours maintainedcollagen matrix and exhibited removal of most cells from the tissue.

FIG. 56 is a series of images demonstrating that decellularized tracheacontained all the three types of COL seen in native trachea.

FIG. 57 is a series of images demonstrating that decellularized tracheasupported NHBE adhesion and growth.

FIG. 58 is a series of images demonstrating that decellularized tracheasupported SAEC adhesion and growth.

FIG. 59 is a series of images demonstrating that NHBE infected with GFPlentivirus did not show obvious morphology change after 6 hours.

FIG. 60 is a series of images demonstrating that significant numbers ofmicrospheres were present in every lobe of the mouse lung followingdelivery by instillation into the airway.

FIG. 61 is a series of images demonstrating the successful injection ofcells into the lungs, and that human epithelial cells that have beentransduced with a transgene (GFP) adhered to lung epithelium.

FIG. 62 is a series of images depicting GFP cells used for injection asseen before trypsinization.

FIGS. 63A through 63C are a series of images demonstrating that that GFPpositive human airway epithelial cells (both NHBE and SAEC) were foundin mouse lungs for days after instillation into the airway.

FIGS. 64A and 64B are a series of images demonstrating the implantedengineered lung at inflation and deflation during the ventilatory cycle.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an engineered lung tissue. The presentinvention is partly based on the discovery that a three dimensional lungtissue can be generated to exhibit characteristics of a natural lungtissue.

In one embodiment, the engineered lung tissue is derived from adecellularized native lung tissue. The decellularized tissues aresubstantially devoid of cells and DNA. Preferably, the decellularizedtissue is also devoid of immunogenic molecules. More preferably, thedecellularized tissue retains several key extracellular matrix moleculesthat are important for cell attachment and proliferation.

The invention includes a method of decellularizing a tissue. Thedecellularization method includes removing cellular and nuclear materialfrom the tissue while retaining key aspects of and minimizing any damageto the extracellular matrix of the lung. In one embodiment, thedecellularization process also includes removing antigenic moleculesfrom the tissue thereby rendering the tissue non-immunogenic. In oneembodiment, the decellularization process of the invention includesgenerating a decellularized scaffold that is fully compatible with cellculture and at the same time provides a barrier function. Preferably,the decellularized scaffold is a lung scaffold that has an intact airwaytree and vascular network.

The invention also includes a bioreactor. Preferably, the bioreactor iscapable of supporting the in vitro culturing of any 3-dimensionaltissue. In one embodiment, the bioreactor is capable of ventilatinglungs via negative pressure as well as providing vascular perfusion andventilation at physiologic rates and pressures. The bioreactor enablesamong other things the perfusion of media through the vasculature, themovement of media or air in and out of the airways, and the ventilationof the lungs via negative (as well as positive) pressure.

The in vitro three dimensional model of lung tissue of the invention isuseful for investigating lung developmental biology. In addition, themodel is useful for among other things, drug discovery, toxicitytesting, disease pathology, and the like.

The invention is also related to the discovery that lung tissue can begenerated in vitro. The in vitro model recapitulates the formation ofstructures reminiscent of alveolar forming units comprised of ductalepithelium tightly interfaced with the host circulation. Accordingly,the invention provides methods and compositions for the generation ofvascularized pulmonary tissues as a form of regenerative medicine.

The invention also provides a method of alleviating or treating a lungdefect in a mammal, preferably a human. The method comprisesadministering to the mammal in need thereof a therapeutically effectiveamount of a composition comprising a three dimensional construct of theinvention, thereby alleviating or treating the lung defect in themammal.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well known and commonly employedin the art.

Standard techniques are used for nucleic acid and peptide synthesis. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references (e.g.,Sambrook and Russell, 2001, Molecular Cloning, A Laboratory Approach,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., andAusubel et al., 2002, Current Protocols in Molecular Biology, John Wiley& Sons, New York, N.Y.), which are provided throughout this document.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent based on the context in which it isused.

The terms “precursor cell,” “progenitor cell,” and “stem cell” are usedinterchangeably in the art and as used herein refer either to apluripotent or lineage-uncommitted progenitor cell, which is potentiallycapable of an unlimited number of mitotic divisions to either renewitself or to produce progeny cells which will differentiate into thedesired cell type. In contrast to pluripotent stem cells,lineage-committed progenitor cells are generally considered to beincapable of giving rise to numerous cell types that phenotypicallydiffer from each other. Instead, progenitor cells give rise to one orpossibly two lineage-committed cell types.

The term “dedifferentiation”, as used herein, refers to the return of acell to a less specialized state. After dedifferentiation, such a cellwill have the capacity to differentiate into more or different celltypes than was possible prior to re-programming. The process of reversedifferentiation (i.e., de-differentiation) is likely more complicatedthan differentiation and requires “re-programming” the cell to becomemore primitive.

As used herein, “scaffold” refers to a structure, comprising abiocompatible material, that provides a surface suitable for adherenceand proliferation of cells. A scaffold may further provide mechanicalstability and support. A scaffold may be in a particular shape or formso as to influence or delimit a three-dimensional shape or form assumedby a population of proliferating cells. Such shapes or forms include,but are not limited to, films (e.g. a form with two-dimensionssubstantially greater than the third dimension), ribbons, cords, sheets,flat discs, cylinders, spheres, 3-dimensional amorphous shapes, etc.

As used here, “biocompatible” refers to any material, which, whenimplanted in a mammal, does not provoke an adverse response in themammal. A biocompatible material, when introduced into an individual, isnot toxic or injurious to that individual, nor does it induceimmunological rejection of the material in the mammal.

As used herein, “autologous” refers to a biological material derivedfrom the same individual into whom the material will later bere-introduced.

As used herein, “allogeneic” refers to a biological material derivedfrom a genetically different individual of the same species as theindividual into whom the material will be introduced.

As used herein, a “graft” refers to a cell, tissue or organ that isimplanted into an individual, typically to replace, correct or otherwiseovercome a defect. A graft may further comprise a scaffold. The tissueor organ may consist of cells that originate from the same individual;this graft is referred to herein by the following interchangeable terms:“autograft”, “autologous transplant”, “autologous implant” and“autologous graft”. A graft comprising cells from a geneticallydifferent individual of the same species is referred to herein by thefollowing interchangeable terms: “allograft”, “allogeneic transplant”,“allogeneic implant” and “allogeneic graft”. A graft from an individualto his identical twin is referred to herein as an “isograft”, a“syngeneic transplant”, a “syngeneic implant” or a “syngeneic graft”. A“xenograft”, “xenogeneic transplant” or “xenogeneic implant” refers to agraft from one individual to another of a different species.

As used herein, the terms “tissue grafting” and “tissue reconstructing”both refer to implanting a graft into an individual to treat oralleviate a tissue defect, such as a lung defect or a soft tissuedefect.

As used herein, to “alleviate” a disease, defect, disorder or conditionmeans reducing the severity of one or more symptoms of the disease,defect, disorder or condition.

As used herein, to “treat” means reducing the frequency with whichsymptoms of a disease, defect, disorder, or adverse condition, and thelike, are experienced by a patient.

As used herein, a “therapeutically effective amount” is the amount of acomposition of the invention sufficient to provide a beneficial effectto the individual to whom the composition is administered.

As used herein, the term “growth medium” is meant to refer to a culturemedium that promotes growth of cells. A growth medium will generallycontain animal serum. In some instances, the growth medium may notcontain animal serum.

“Differentiation medium” is used herein to refer to a cell growth mediumcomprising an additive or a lack of an additive such that a stem cell,fetal pulmonary cell or other such progenitor cell, that is not fullydifferentiated, develops into a cell with some or all of thecharacteristics of a differentiated cell when incubated in the medium.

As used herein, the term “growth factor product” refers to a protein,peptide, mitogen, or other molecule having a growth, proliferative,differentiative, or trophic effect on a cell. Growth factors include,but are not limited to, fibroblast growth factor (FGF), basic fibroblastgrowth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermalgrowth factor (EGF), insulin-like growth factor-I (IGF-T), insulin-likegrowth factor-II (IGF-II), platelet-derived growth factor (PDGF),vascular endothelial cell growth factor (VEGF), activin-A, bonemorphogenic proteins (BMPs), insulin, growth hormone, erythropoietin,thrombopoietin, interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin7 (IL-7), macrophage colony stimulating factor, c-kit ligand/stem cellfactor, osteoprotegerin ligand, insulin, nerve growth factor, ciliaryneurotrophic factor, cytokines, chemokines, morphogens, neutralizingantibodies, other proteins, and small molecules. Preferably, the FGF isselected from the group selected from FGF2, FGF7, FGF10, and anycombination thereof.

An “isolated cell” refers to a cell which has been separated from othercomponents and/or cells which naturally accompany the isolated cell in atissue or mammal.

As used herein, a “fetal pulmonary cells” (FPCs) refer to cells isolatedfrom the lung tissue of an embryo. A mixed population of FPCs caninclude, but is not limited to epithelial, mesenchymal, and endothelialcells.

As used herein, “epithelial cell” means a cell which forms the outersurface of the body and lines organs, cavities and mucosal surfaces.

As used herein, “endothelial cell” means a cell which lines the bloodand lymphatic vessels and various other body cavities.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. Thus, a substantially purifiedcell refers to a cell which has been purified from other cell types withwhich it is normally associated in its naturally-occurring state.

“Expandability” is used herein to refer to the capacity of a cell toproliferate, for example, to expand in number or, in the case of apopulation of cells, to undergo population doublings.

The term “lung specific” refers to a nucleic acid molecule orpolypeptide that is expressed predominantly in the lung as compared toother tissues in the body. In a preferred embodiment, a “lung specific”nucleic acid molecule or polypeptide is expressed at a level that is5-fold higher than any other tissue in the body. In a more preferredembodiment, the “lung specific” nucleic acid molecule or polypeptide isexpressed at a level that is 10-fold higher than any other tissue in thebody, more preferably at least 15-fold, 20-fold, 25-fold, 50-fold or100-fold higher than any other tissue in the body. Nucleic acid moleculelevels may be measured by nucleic acid hybridization, such as Northernblot hybridization, or quantitative PCR. Polypeptide levels may bemeasured by any method known to accurately measure protein levels, suchas Western blot analysis.

“Proliferation” is used herein to refer to the reproduction ormultiplication of similar forms, especially of cells. That is,proliferation encompasses production of a greater number of cells, andcan be measured by, among other things, simply counting the numbers ofcells, measuring incorporation of ³H-thymidine into the cell, and thelike.

As used herein, “tissue engineering” refers to the process of generatingtissues ex vivo for use in tissue replacement or reconstruction. Tissueengineering is an example of “regenerative medicine,” which encompassesapproaches to the repair or replacement of tissues and organs byincorporation of cells, gene or other biological building blocks, alongwith bioengineered materials and technologies.

As used herein “endogenous” refers to any material from or producedinside an organism, cell or system.

“Exogenous” refers to any material introduced into or produced outsidean organism, cell, or system.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in anaturally-occurring state, i.e., a DNA fragment which has been removedfrom the sequences which are normally adjacent to the fragment, i.e.,the sequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to the polynucleotides to control RNA polymeraseinitiation and expression of the polynucleotides.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

The term “tissue,” as used herein includes, but is not limited to, bone,neural tissue, fibrous connective tissue including tendons andligaments, cartilage, dura, pericardia, muscle, lung, heart valves,veins and arteries and other vasculature, dermis, adipose tissue, orglandular tissue.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a cell substantiallyonly if the cell is a cell of the tissue type corresponding to thepromoter.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (i.e., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) anda primate (e.g., monkey and human), most preferably a human.

Description

The present invention provides an engineered three dimensional pulmonarytissue and methods of making the three dimensional pulmonary tissue.Preferably, the pulmonary tissue is a lung tissue. In one embodiment,the engineered pulmonary tissue exhibits branching morphogenesisexemplified by natural pulmonary tissue. Thus, the invention provides anin vitro model that mimics natural pulmonary tissue. The in vitro threedimensional pulmonary tissue model is useful for among other things,drug discovery, toxicity testing, disease pathology, and the like.

The invention is based on the discovery of a procedure useful fordecellularizing lung tissue using a technique that removes cellularmaterial but that retains key components of the extracellular matrix.The development of a decellularized lung matrix is important as ascaffold for tissue engineering applications. Accordingly, the inventionincludes a method for substantially decellularizing a tissue or organ.Preferably, the method significantly reduces or eliminatesimmunogenicity of the tissue or organ such that upon transplantation,the tissue or organ is not rejected by the recipient's immune system.The method includes removing the tissue from a donor, processing thetissue to remove substantially all of the cells of the tissue or organ.The method further includes repopulating the decellularized scaffoldthrough seeding with cells including but not limited to stem cells,fetal cells and the like, for implantation into recipient. Preferably,the decellularized scaffold is seeded with non-immunogenic cells. In oneembodiment, the decellularized scaffold is seeded with cells that areautologous to the intended recipient. Depending on the type of tissuebeing treated and to be replaced, different stem cells known in the artor which become known hereafter are selected such that appropriatetissues are formed upon implantation into a recipient of the seededimplant.

In some instances, the engineered three dimensional pulmonary tissuecomprises cells cultured on the tissue. Any suitable cells can be usedfor culturing on the decellularized tissue of the invention. In someinstances, stems cells are cultured on the decellularized tissue forregeneration of lung tissue. In some instances, fetal or neonatalpulmonary cells (NPCs) are cultured on the decellularized tissue. Insome instances, a mixed population of NPCs are used, wherein thepopulation of NPCs include, but are not limited to epithelial cells,mesenchymal cells, and endothelial cells.

After seeding, the cells on the scaffold are optionally subjected to anexpansion medium or to a differentiation medium or cultured in thepresence of tissue-specific growth factors. The composition is thenimplanted into a subject in need thereof. The subject may be a mammal,but is preferably a human and the source of the cells for growth andimplantation is any mammal, preferably a human. The implantedcomposition supports additional cell growth in vivo, thus providingtissue reconstruction. Accordingly, the invention provides the use ofengineered three dimensional pulmonary tissue for tissue graftingtherapies.

The invention also includes generation of pulmonary tissue in vivo.Preferably, vascularized pulmonary tissue is generated in vivo. In oneaspect, the fetal pulmonary cells are administered in the context of thedecellularized tissue to a mammal to facilitate in vivo pulmonary tissueformation.

In the present invention, it is demonstrated that the decellularizedtissue can be seeded with suitable cells, such as neonatal or adultpulmonary cells, and the resultant composition can be used as avascularized three dimensional pulmonary tissue model for preclinical invitro pharmacological, physiological, and scientific testing. Inaddition, the decellularized tissue can be seeded with suitable cells,such as neonatal pulmonary cells or autologous pulmonary cells, and theresultant composition can be used for tissue reconstruction in vivo.

The compositions and methods of the instant invention have myriad usefulapplications. The compositions may be used in therapeutic methods foralleviating or treating tissue defects in an individual. Thecompositions may also be used in vitro or in vivo to identifytherapeutic compounds and therefore may have therapeutic potential.

Decellularization

The present invention provides an advancement over tissue engineeringtechniques known in the art. Specifically, the present inventionprovides a method of making engineered tissue scaffolds using adecellularized tissue as a starting source, preferably a decellularizednatural tissue derived from a mammal.

The decellularization process relies on a chemical methodology. In oneaspect, the chemical solution or otherwise referred to as thedecellularization solution used for decellularization generally includesat least a hypertonic solution, a detergent, and a chelating agent.Preferably, the hypertonic solution is a hypertonic sodium chloridesolution. Preferably, the detergent is a zwitterionic detergent such asCHAPS. Preferably, the chelating agent is EDTA.

In one embodiment, the decellularization solution can include a buffer(e.g., PBS) for osmotic compatibility with the cells. In some instances,the decellularization solution also can include enzymes such as, withoutlimitation, one or more collagenases, one or more dispases, one or moreDNases, or a protease such as trypsin. In some instances, thedecellularization solution also or alternatively can include inhibitorsof one or more enzymes (e.g., protease inhibitors, nuclease inhibitors,and/or collegenase inhibitors).

In one embodiment, the method to decellularize a tissue of the inventionincludes perfusing the tissue with the decellularization solution. Thepressure for which the decellularization solution is perfused throughthe tissue can be adjusted to the desired pressure. Preferably, thedecellularization solution is perfused through the tissue at perfusionpressure below about 30 mmHg. More preferably, the decellularizationsolution is perfused through the tissue at pressures less than about 20mmHg.

In one embodiment, the decellularization solution can be introduced intothe airway of the lung tissue to effect cell removal.

In one embodiment, the decellularized tissue of the invention consistsessentially of the extracellular matrix (ECM) component of all or mostregions of the tissue, including ECM components of the vascular tree.ECM components can include any or all of the following: fibronectin,fibrillin, laminin, elastin, members of the collagen family (e.g.,collagen I, III, and IV), glycosaminoglycans, ground substance,reticular fibers and thrombospondin, which can remain organized asdefined structures such as the basal lamina. Successfuldecellularization is defined as the absence of detectable myofilaments,endothelial cells, smooth muscle cells, epithelial cells, and nuclei inhistologic sections using standard histological staining procedures.Preferably, but not necessarily, residual cell debris also has beenremoved from the decellularized tissue.

In one embodiment, the decellularization process of a natural tissuepreserves the native 3-dimensional structure of the tissue. That is, themorphology and the architecture of the tissue, including ECM componentsbe maintained during and following the process of decellularization. Themorphology and architecture of the ECM can be examined visually and/orhistologically. For example, the basal lamina on the exterior surface ofa solid organ or within the vasculature of an organ or tissue should notbe removed or significantly damaged due to decellularization. Inaddition, the fibrils of the ECM should be similar to or significantlyunchanged from that of an organ or tissue that has not beendecellularized.

In one embodiment, one or more compounds can be applied in or on adecellularized tissue to, for example, preserve the decellularizedtissue, or to prepare the decellularized tissue for recellularizationand/or to assist or stimulate cells during the recellularizationprocess. Such compounds include, but are not limited to, one or moregrowth factors (e.g., VEGF, DKK-1, FGF, BMP-1, BMP-4, SDF-1, IGF, andHGF), immune modulating agents (e.g., cytokines, glucocorticoids, IL2Rantagonist, leucotriene antagonists), and/or factors that modify thecoagulation cascade (e.g., aspirin, heparin-binding proteins, andheparin). In addition, a decellularized organ or tissue can be furthertreated with, for example, irradiation (e.g., UV, gamma) to reduce oreliminate the presence of any type of microorganism remaining on or in adecellularized tissue.

Use of the decellularization solution of the invention to generate adecellularized tissue provides a controlled, precise way to destroycells of a tissue, while leaving the underlying ECM, includingvascularization, and other gross morphological features of the originaltissue intact. The decellularized scaffolds are then suitable forseeding with appropriate cells. Where the process is performed in vitro,the seeded tissue is suitable for implantation into the recipient as areplacement tissue. In addition to the decellularized tissuesthemselves, the invention includes methods of fabrication of engineeredtissues built from such scaffolds.

The present invention provides a method suitable for producing a tissuescaffold for use in tissue engineering. Although the source of thetissue is not limited, in exemplary embodiments, the tissue is from arelatively large animal or an animal recognized as having a similaranatomy (with regard to the tissue of interest) as a human, such as apig, a cow, a horse, a monkey, or an ape. In some embodiments, thesource of the tissue is human, use of which can reduce the possibilityof rejection of engineered tissues based on the scaffold. In preferredembodiments, the method leaves intact vascular structures of the tissue,such as alveolar architecture with preservation of the alveolar septae.As used herein, the term “intact” refers to a state of being whereby anelement is capable of performing its original function to a substantialextent.

In one embodiment, the decellularized lung retains several keycharacteristics of normal lung matrix. For example, the decellularizedlung comprises at least one or more of collagen, elastin, fibronectin,and proteoglycan

The decellularized tissue does not retain either majorhistocompatibility complex (MHC) class I or II antigen, therefore thetissue does not elicit an adverse an immune response when administeredto a recipient.

The decellularized tissue retains mechanics properties of normal nativelung. The decellularized tissue also retains some of the barrierfunction of normal native lung.

Bioreactor

The invention provides a system (e.g., a bioreactor) for decellularizingand/or recellularizing tissue. The bioreactor enables the maintenance ofcell viability, cellular differentiation state, and lung morphology.Decellularized scaffolds, when cultured in the bioreactor with asuitable cell source, can support the adherence and proliferation of awide range of cell types, including pulmonary endothelial, epithelial,and mesenchymal cells. The bioreactor of the invention incorporates keyfeatures of the vivo environment. The bioreactor was designed to allowmodifications for optimizing decellularization and/or recellularizationprocesses. In one embodiment, the bioreactor is capable of perfusingmedia through the vasculature at a rate specified by the user and withinthe physiological flow and pressure levels of a mammal. In anotherembodiment, the bioreactor is capable of ventilating the tissue (e.g.,lung) with air or media through the trachea. Preferably, negativepressure ventilation is used in order to be consistent with normalphysiological conditions, though ventilation using positive pressure canalso be done. In yet another embodiment, the bioreactor is capable ofallowing different media types to bathe the vascular and airwaycompartments of the tissue. In another embodiment, the bioreactor allowsfor gas exchange into the culture medium, while simultaneously meetingthe desired requirements for ventilation. In another embodiment, thebioreactor has ports to allow for pressure measurements, for examplemeasurements of the pulmonary artery and tracheal pressures. Preferably,pressures are within normal physiological values. In another embodiment,the bioreactor has a means of allowing media exchange on a periodicbasis.

The bioreactor of the invention generally includes at least onecannulation device for cannulating a tissue, a perfusion apparatus forperfusing media through the cannula(s), and means (e.g., a containmentsystem) to maintain a sterile environment for the organ or tissue. Acannulation device generally includes size-appropriate hollow tubing forintroducing into a vessel, duct, and/or cavity of a tissue. Typically,one or more vessels, ducts, and/or cavities are cannulated in a tissue.A perfusion apparatus can include a holding container for the liquid(e.g., a cellular disruption medium) and a mechanism for moving theliquid through the organ (e.g., a pump, air pressure, gravity) via theone or more cannulae. The sterility of a tissue during decellularizationand/or recellularization can be maintained using the methods discussedelsewhere herein.

The bioreactor for can be used to decellularize and recellularizetissues as described herein. The process can be monitored for certainperfusion characteristics (e.g., pressure, volume, flow pattern,temperature, gases, pH), mechanical forces (e.g., ventricular wallmotion and stress), and electrical stimulation (e.g., pacing). Theeffectiveness of perfusion can be evaluated in the effluent and intissue sections. Perfusion volume, flow pattern, temperature, partial O₂and CO₂ pressures and pH can be monitored using standard methods.

Sensors can be used to monitor the bioreactor and/or the tissue.Sonomicromentry, micromanometry, and/or conductance measurements can beused to acquire pressure-volume. For example, sensors can be used tomonitor the pressure of a liquid moving through a cannulated organ ortissue; the ambient temperature in the system and/or the temperature ofthe organ or tissue; the pH and/or the rate of flow of a liquid movingthrough the cannulated organ or tissue; and/or the biological activityof a recellularizing tissue. In addition to having sensors formonitoring such features, a system for decellularizing and/orrecellularizing a tissue also can include means for maintaining oradjusting such features. Means for maintaining or adjusting suchfeatures can include components such as a thermometer, a thermostat,electrodes, pressure sensors, overflow valves, valves for changing therate of flow of a liquid, valves for opening and closing fluidconnections to solutions used for changing the pH of a solution, aballoon, an external pacemaker, and/or a compliance chamber. To helpensure stable conditions (e.g., temperature), the chambers, reservoirsand tubings can be water-jacketed.

The bioreactor is capable of providing sufficient nutrient supply andmechanical stimulation to the lung tissue in order to support cellsurvival and differentiation. The bioreactor can be used for in vitrolung tissue culture and for engineered lung tissue culture. Preferably,the bioreactor is used to culture engineered lung tissue using thedecellularized lung scaffolds of the invention.

The development of a bioreactor capable of the in vitro culture of true3-dimensional segments of lung tissue is an important step in thedevelopment of clinically useful engineered lung tissue. For example,growth and maturation of the engineered lung tissue can take place inthe bioreactor prior to implantation of the engineered lung into arecipient, thereby enhancing the functionality of the final implantedlung tissue in vivo. In addition, the bioreactor for in vitro lungculture can be used to assist the study of pulmonary biology,physiology, and development. That is, the interactions of lungendothelial and epithelial cells to form the alveolar-capillary barriercan be studied using the engineered lung tissue and bioreactor of theinvention. A skilled artisan would be able to study lung behavior in amore controlled environment than the various animal models currentlyused. The engineered lung tissue and bioreactor could also be used forpharmacologic testing and investigation in human or animal tissue beforeproceeding to time-consuming and costly human or animal trials.

Compositions

Compositions of the invention include an engineered lung tissue.Preferably, the engineered lung tissue exhibits any one or more of thefollowing properties: 1) vasculature and airway, where there is apatent, perfused vasculature and a patent airway tree that can beventilated; 2) gas exchange, where the engineered lung is capable ofexchanging sufficient gas between the airway and vascular compartmentsto support the physiological needs of the recipient; most preferably,the partial pressure of oxygen in the pulmonary vein is at least 50mmHg; 3) mechanics, where the engineered tissue is strong enough towithstand all needed movements, in particular breathing motions andvascular perfusion, as well as manipulation during surgicalimplantation; 4) immunogenicity, where the engineered lung tissue doesnot provoke an immune response when implanted into the recipient.

The compositions and methods of the instant invention can be practicedusing any suitable cell. Preferably, the suitable cell or cells areregenerative and can be used to recellularize the decellularized tissueof the invention. An example of a regenerative cells includes, but isnot limited to, a stem cell, an embryonic stem cell, an adult stem cell,an umbilical cord blood cell, a tissue-derived stem or progenitor cells,bone marrow-derived step or progenitor cells, blood-derived stem orprogenitor cell, a mesenchymal stem cells (MSC), a skeletalmuscle-derived cells, a multipotent adult progentitor cell (MAPC), afetal pulmonary cell, differentiated pulmonary epithelial cells,pulmonary progenitor cells, vascular progenitor cells, differentiatedvascular cells and the like. Additional regenerative cells that can beused include bone marrow-derived stem cells such as bone marrowmononuclear cells (BM-MNC), endothelial or vascular stem or progenitorcells, and peripheral blood-derived stem cells such as endothelialprogenitor cells (EPC).

Preferably, the suitable cell is isolated from a mammal, more preferablya primate and more preferably still, a human. The cells useful in themethods of the present invention are isolated using methods discussedherein, for example in the Examples section, or by any method known inthe art. Following isolation, the suitable cells are cultured in aculture medium.

As a non-limiting example, neonatal pulmonary cells (NPCs) are describedin more detailed with respect to culturing the cells. However, a skilledartisan will recognize that the culturing conditions can be modified tothe suitable cell. Media formulations that support the growth ofpulmonary cells include, but are not limited to, Minimum EssentialMedium Eagle, ADC-1, LPM (bovine serum albumin-free), F10 (HAM), F12(HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and withoutFitton-Jackson Modification), Basal Medium Eagle (BME—with the additionof Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM—withoutserum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM),Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E—withEarle's salt base), Medium M199 (M199H—with Hank's salt base), MinimumEssential Medium Eagle (MEM-E—with Earle's salt base), Minimum EssentialMedium Eagle (MEM-H—with Hank's salt base) and Minimum Essential MediumEagle (MEM-NAA with nonessential amino acids), and the like.

Additional non-limiting examples of media useful in the methods of theinvention may contain fetal serum of bovine or other species at aconcentration at least 1% to about 30%, preferably at least about 5% to15%, most preferably about 10%. Embryonic extract of bovine or otherspecies can be present at a concentration of about 1% to 30%, preferablyat least about 5% to 15%, most preferably about 10%.

Typically, the NPC culture medium comprises a base medium, serum and anantibiotic/antimycotic. One preferred base medium is DMEM/F12 (1:1). Thepreferred serum is fetal bovine serum (FBS) but other sera may be used,including horse serum or human serum. Preferably up to 20% FBS will beadded to the above medium in order to support the growth of NPCs.However, a defined medium can be used if the necessary growth factors,cytokines, and hormones in FBS for NPC growth are identified andprovided at appropriate concentrations in the growth medium. It isfurther recognized that additional components may be added to theculture medium. Such components include, but are not limited to,antibiotics, antimycotics, albumin, growth factors, amino acids, andother components known to the art for the culture of cells. Antibioticswhich can be added into the medium include, but are not limited to,penicillin and streptomycin. The concentration of penicillin in theculture medium is about 10 to about 200 units per ml. The concentrationof streptomycin in the culture medium is about 10 to about 200 μg/ml.However, the invention should in no way be construed to be limited toany one medium for culturing NPCs. Rather, any media capable ofsupporting pulmonary cells in tissue culture may be used.

In addition, the NPC culture medium can be supplemented with at leastone growth factor. Preferably the growth factor is fibroblast growthfactor (FGF). For example, any combination of FGF10, FGF7, FGF2 can besupplemented to the NPC culture medium. A preferred concentration ofFGF7 is about 0.1-100 ng/ml (and any integer in between), morepreferably the concentration is about 10 ng/ml. A preferredconcentration of FGF10 is about 1-200 ng/ml (and any integer inbetween), more preferably the concentration is about 25 ng/ml. Apreferred concentration of FGF2 is about 1-200 ng/ml (and any integer inbetween), more preferably the concentration is about 25 ng/ml.

Following isolation, NPCs may be incubated in culture medium, in aculture apparatus for a period of time or until the cells reachconfluency before passing the cells to another culture apparatus.Following the initial plating, the cells can be maintained in culturefor a period of about 6 days to yield the Passage 0 (P0) population. Thecells may be passaged for an indefinite number of times, each passagecomprising culturing the cells for about 6-7 days, during which time thecell doubling time can range between about 3 to about 5 days. Theculturing apparatus can be of any culture apparatus commonly used inculturing cells in vitro.

NPCs may be cultured in culture medium supplemented with FGF in the fora period of time or until the cells reach a certain level of confluence.Preferably, the level of confluence is greater than 70%. Morepreferably, the level of confluence is greater than 90%. A period oftime can be any time suitable for the culture of cells in vitro. NPCculture medium may be replaced during the culture of NPCs at any time.Preferably, the culture medium is replaced every 3 to 4 days. NPCs arethen harvested from the culture apparatus whereupon they may be usedimmediately or cryopreserved to be stored for use at a later time. NPCsmay be harvested by trypsinization, EDTA treatment, or any otherprocedure used to harvest cells from a culture apparatus.

NPCs described herein may be cryopreserved according to routineprocedures. Preferably, about one to ten million cells are cryopreservedin culture medium containing 10% DMSO in vapor phase of liquid N₂.Frozen cells may be thawed by swirling in a 37° C. bath, resuspended infresh growth medium, and expanded as described above.

The invention also provides cells that “seed” the scaffold. NPCs can becultured on the scaffold. The cells can also differentiate in vitro byculturing the cells in differentiation medium. Alternatively, the cellscan differentiate in vivo when they establish contact with a tissuewithin the mammal or when the cells are sufficiently close to a tissueto be influenced by substances (e.g., growth factors, enzymes, orhormones) released from the tissue. In other words, NPCs of the matrixcan establish contact with a tissue, such as lung, by virtue ofreceiving signals from the tissue. Such signaling would occur, forexample, when a receptor on the surface of a NPC, or on the surface of acell descended from a NPC, binds and transduces a signal from a moleculesuch as a growth factor, enzyme, or hormone that was released by atissue within the mammal. These agents guide differentiation so that theNPCs come to express some and possibly most (if not all) of the sameproteins normally expressed by differentiated cells in the tissue inwhich they have been placed.

Alternatively, or in addition, NPCs of the matrix can be induced todifferentiate by adding a substance (e.g., a growth factor, enzyme,hormone, or other signaling molecule) to the cell's environment. Forexample, a substance can be added to the biological scaffolding of theinvention.

While NPCs and associated cellular matrix can eventually become fullydifferentiated, and while this is desirable in some circumstances (e.g.,where the cells are used to recreate a histologically mature andcomplete tissue), not all of the cells administered need to be fullydifferentiated to achieve successful treatment; NPCs of the cellularmatrix need only differentiate to a point sufficient to treat themammal. That point can be reached either before or after the matrix isadministered to the patient.

Differentiation occurs when a cell of the matrix expresses essentiallythe same phenotype as a mature cell at the site of implantation. Forexample, for the purpose of defining this invention, a NPC of a cellularmatrix, having been implanted into the lung, is differentiated when itexpresses essentially the same proteins expressed by the lung, e.g., analveolar epithelial cell. Antibodies to lung markers are commerciallyavailable or otherwise readily attainable.

Differentiated cells can also be identified by their gross morphologyand by the connections they form with other cells. For example, cellsthat differentiate into lung cells can develop complex morphologyresembling bronchioles. For example, the invention is based on the noveldiscovery that culturing NPCs on a three dimensional scaffold canexhibit characteristics of mature lung cells.

The number of cells that is introduced into and onto a decellularizedorgan in order to generate an organ or tissue is dependent on both theorgan (e.g., which organ, the size and weight of the organ) or tissueand the type and developmental stage of the regenerative cells.Different types of cells may have different tendencies as to thepopulation density those cells will reach. Similarly, different organ ortissues may be cellularized at different densities. By way of example, adecellularized organ or tissue can be seeded with at least about 1,000(e.g., at least 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000)regenerative cells; or can have from about 1,000 cells/mg tissue (wetweight, i.e., prior to decellularization) to about 10,000,000 cells/mgtissue (wet weight) attached thereto.

Cells can be introduced to a decellularized organ or tissue by injectioninto one or more locations. In addition, more than one type of cell(i.e., a cocktail of cells) can be introduced into a decellularizedorgan or tissue. For example, a cocktail of cells can be injected atmultiple positions in a decellularized organ or tissue or different celltypes can be injected into different portions of a decellularized organor tissue. Alternatively, or in addition to injection, regenerativecells or a cocktail of cells can be introduced by perfusion into acannulated decellularized organ or tissue. For example, cells can beperfused into a decellularized organ using a perfusion medium, which canthen be changed to an expansion and/or differentiation medium to inducegrowth and/or differentiation of the regenerative cells. In the case ofa lung tissue, the cells can be introducted into either or both of theairway compartment via the trachea, or the vascular compartment via thepulmonary artery or vein.

During recellularization, an organ or tissue is maintained underconditions in which at least some of the regenerative cells can multiplyand/or differentiate within and on the decellularized organ or tissue.Those conditions include, without limitation, the appropriatetemperature and/or pressure, electrical and/or mechanical activity,force, the appropriate amounts of O₂ and/or CO₂, an appropriate amountof humidity, and sterile or near-sterile conditions. Duringrecellularization, the decellularized organ or tissue and the cellsattached thereto are maintained in a suitable environment. For example,the cells may require a nutritional supplement (e.g., nutrients and/or acarbon source such as glucose), exogenous hormones or growth factors,and/or a particular pH.

Cells can be allogeneic to a decellularized organ or tissue (e.g., ahuman decellularized organ or tissue seeded with human cells), orregenerative cells can be xenogeneic to a decellularized organ or tissue(e.g., a pig decellularized organ or tissue seeded with human cells).

In some instances, an organ or tissue generated by the methods describedherein is to be transplanted into a patient. In those cases, the cellsused to recellularize a decellularized organ or tissue can be obtainedfrom the patient such that the regenerative cells are autologous to thepatient. Cells from a patient can be obtained from, for example, blood,bone marrow, tissues, or organs at different stages of life (e.g.,prenatally, neonatally or perinatally, during adolescence, or as anadult) using methods known in the art. Alternatively, cells used torecellularize a decellularized organ or tissue can be syngeneic (i.e.,from an identical twin) to the patient, cells can be human lymphocyteantigen (HLA)-matched cells from, for example, a relative of the patientor an HLA-matched individual unrelated to the patient, or cells can beallogeneic to the patient from, for example, a non-HLA-matched donor.

Irrespective of the source of the cells (e.g., autologous or not), thedecellularized solid organ can be autologous, allogeneic or xenogeneicto a patient.

In certain instances, a decellularized tissue may be recellularized withcells in vivo (e.g., after the tissue has been transplanted into anindividual). In vivo recellularization may be performed as describedabove (e.g., injection and/or perfusion) with, for example, any of thecells described herein. Alternatively or additionally, in vivo seedingof a decellularized organ or tissue with endogenous cells may occurnaturally or be mediated by factors delivered to the recellularizedtissue.

Genetic Modification

The present invention relates to the discovery that the decellularizedtissues of the invention can be used to facilitate lung cell therapy ina mammal.

In another embodiment, decellularized lung tissue can be used to culturedesired lung cells such as pulmonary epithelial cells. Whethergenetically modified or not, the cells can be used to treat a lungdisease including but not limited to emphysema, bronchiolitisobliterans, and cystic fibrosis. For example, the decellularized tissueof the invention can be used as a substrate for the culture of humanpulmonary airway epithelial cells. The cultured human airway epithelialcells can then be delivered to a recipient via tracheal instillation,inhalation, or injection, among other ways. Such cells that are expandedin culture can be used to effect therapy in the recipient. Thedecellularized lung tissue (e.g., trachea) provides an outstandingplatform for culturing and expanding the pulmonary epithelial cells,which are normally very difficult to grow in typical cell cultureenvironment, such as tissue culture plastic.

In the context of gene therapy, the cells cultured on the decellularizedtissue can be treated with a gene of interest prior to delivery of thecells into the lung of a recipient. In some cases, such cell-based genedelivery can present significant advantages of other means of genedelivery to the lung, such as inhalation of adenoviral gene deliveryvectors. This superiority of cell-based gene delivery to a host stemsfrom the observation that inhaled gene delivery vectors typically resultin poor efficiency of cellular transduction, due to barriers imposed bythe mucous layer and the host immune system. Delivery of a therapeuticgene that has been pre-inserted into cells avoids the problemsassociated with penetration of gene therapy vectors into recipient lungcells.

The decellularized lung tissue of the invention provides a convenientand efficient means to grow lung cells such as epithelial cells in ahighly viable and differentiated state, as compared to culture onstandard tissue culture plastic. In turn, the expansion of lung cellssuch as pulmonary epithelial cells on the decellularized matrix providesfor a sufficiently large number of cells to be efficacious for celltherapy. In addition, the expansion of lung epithelial cells on thedecellularized matrix provides a platform whereby cultured cells and betreated with gene therapy vectors in vitro. Cells that are transfectedwith a gene of choice in vitro can them be optionally purified to selectfor only those cells expressing the transgene of interest, and thenintroduced into a recipient in need of such cellular therapy. Such anapproach could be of particular value in treating genetic lung diseasessuch as cystic fibrosis.

In one embodiment, the invention provides a method of treating cysticfibrosis. The method includes transfecting cells of interest such asepithelial cells with a normal version of the CFTR gene, a mutatedversion of which is the gene responsible for cystic fibrosis. Deliveryof such transfected cells into a patient, either by instillation intothe trachea, inhalation, or other means of introduction, alleviates thesignificant difficulties that have been associated with delivery of genevectors into these patients. In this way, efficacious cellular therapyand gene delivery in cystic fibrosis may be realized. However, theinvention should not be limited to only treating cystic fibrosis withcells transfected with the CFTR gene. Rather, the invention includes thetreatment of any disease or disorder associated with lung cells.

Accordingly, the invention provides the use of genetically modifiedcells, such as pulmonary cells, that have been cultured on thedecellularized tissue of the invention. Genetic modification may, forinstance, result in the expression of exogenous genes (“transgenes”) orin a change of expression of an endogenous gene. Such geneticmodification may have therapeutic benefit. Alternatively, the geneticmodification may provide a means to track or identify the cellsso-modified, for instance, after implantation of a composition of theinvention into an individual. Tracking a cell may include trackingmigration, assimilation and survival of a transplantedgenetically-modified cell. Genetic modification may also include atleast a second gene. A second gene may encode, for instance, aselectable antibiotic-resistance gene or another selectable marker.

Proteins useful for tracking a cell include, but are not limited to,green fluorescent protein (GFP), any of the other fluorescent proteins(e.g., enhanced green, cyan, yellow, blue and red fluorescent proteins;Clontech, Palo Alto, Calif.), or other tag proteins (e.g., LacZ,FLAG-tag, Myc, His₆, and the like).

When the purpose of genetic modification of the cell is for theproduction of a biologically active substance, the substance willgenerally be one that is useful for the treatment of a given disorder.For example, it may be desired to genetically modify cells so that theysecrete a certain growth factor product associated with bone or softtissue formation. Growth factor products to induce growth of other,endogenous cell types relevant to tissue repair are also useful. Forinstance, growth factors to stimulate endogenous capillary and/ormicrovascular endothelial cells can be useful in repair of soft tissuedefect, especially for larger volume defects.

The cells of the present invention can be genetically modified by havingexogenous genetic material introduced into the cells, to produce amolecule such as a trophic factor, a growth factor, a cytokine, and thelike, which is beneficial to culturing the cells. In addition, by havingthe cells genetically modified to produce such a molecule, the cell canprovide an additional therapeutic effect to the mammal when transplantedinto a mammal in need thereof. For example, the genetically modifiedcell can secrete a molecule that is beneficial to cells neighboring thetransplant site in the mammal.

The pulmonary cells may be genetically modified using any method knownto the skilled artisan. See, for instance, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.), and in Ausubel et al., Eds, (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y.). For example, a pulmonary cell may be exposed to an expressionvector comprising a nucleic acid including a transgene, such that thenucleic acid is introduced into the cell under conditions appropriatefor the transgene to be expressed within the cell. The transgenegenerally is an expression cassette, including a polynucleotide operablylinked to a suitable promoter. The polynucleotide can encode a protein,or it can encode biologically active RNA (e.g., antisense RNA or aribozyme). Thus, for example, the polynucleotide can encode a geneconferring resistance to a toxin, a hormone (such as peptide growthhormones, hormone releasing factors, sex hormones, adrenocorticotrophichormones, cytokines (e.g., interferins, interleukins, lymphokines),etc.), a cell-surface-bound intracellular signaling moiety (e.g., celladhesion molecules, hormone receptors, etc.), a factor promoting a givenlineage of differentiation (e.g., bone morphogenic protein (BMP)), etc.

Within the expression cassette, the coding polynucleotide is operablylinked to a suitable promoter. Examples of suitable promoters includeprokaryotic promoters and viral promoters (e.g., retroviral ITRs, LTRs,immediate early viral promoters (IEp), such as herpesvirus IEp (e.g.,ICP4-IEp and ICP0-IEEp), cytomegalovirus (CMV) IEp, and other viralpromoters, such as Rous Sarcoma Virus (RSV) promoters, and MurineLeukemia Virus (MLV) promoters). Other suitable promoters are eukaryoticpromoters, such as enhancers (e.g., the rabbit .beta.-globin regulatoryelements), constitutively active promoters (e.g., the .beta.-actinpromoter, etc.), signal specific promoters (e.g., inducible promoterssuch as a promoter responsive to RU486, etc.), and tissue-specificpromoters. It is well within the skill of the art to select a promotersuitable for driving gene expression in a predefined cellular context.The expression cassette can include more than one coding polynucleotide,and it can include other elements (e.g., polyadenylation sequences,sequences encoding a membrane-insertion signal or a secretion leader,ribosome entry sequences, transcriptional regulatory elements (e.g.,enhancers, silencers, etc.), and the like), as desired.

The expression cassette containing the transgene should be incorporatedinto a genetic vector suitable for delivering the transgene to thecells. Depending on the desired end application, any such vector can beso employed to genetically modify the cells (e.g., plasmids, naked DNA,viruses such as adenovirus, adeno-associated virus, herpesviruses,lentiviruses, papillomaviruses, retroviruses, etc.). Any method ofconstructing the desired expression cassette within such vectors can beemployed, many of which are well known in the art (e.g., direct cloning,homologous recombination, etc.). The choice of vector will largelydetermine the method used to introduce the vector into the cells (e.g.,by protoplast fusion, calcium-phosphate precipitation, gene gun,electroporation, DEAE dextran or lipid carrier mediated transfection,infection with viral vectors, etc.), which are generally known in theart.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR), the ligase chain reaction (LCR), and other DNA or RNApolymerase-mediated techniques are found in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, volumes 1-3 (3rd ed., Cold Spring HarborPress, NY 2001).

Once the nucleic acid for a protein is cloned, a skilled artisan mayexpress the recombinant gene(s) in a variety of lung cells. It isexpected that those of skill in the art are knowledgeable in thenumerous expression systems available for expressing the desiredtransgene.

Present invention provides a engineered three dimensional tissue thatmimics natural lung tissue. The capability to create composites andscaffolds that mimic natural lung tissue enables the repair andregeneration of tissues and collections of tissues to a greater degreethan prior art methods, and exhibits more accurate histologicalstructure and function than can be achieved using prior art methods. Forexample, the engineered lung tissue comprises cells that exhibit buddingstructures and elongating tubular structures. Furthermore, the cellsexpress genes involved in morphogenesis and lung epithelialdifferentiation. Non-limiting genes involved in morphogenesis and lungepithelial differentiation include distal epithelial marker genes SpCand SpB, the mesenchymal-derived morphogen FGF10, FGFr2, and vascularendothelial growth factor A (VEGF).

Administration

The invention contemplates use of the engineered tissues in both invitro and in vivo settings. Thus, the invention provides for use of theengineered tissues for research purposes and for therapeutic ormedical/veterinary purposes. In research settings, an enormous number ofpractical applications exist for the technology. One example of suchapplications is use of the engineered tissues in an ex vivo cancermodel, such as one to test the effectiveness of various ablationtechniques (including, for example, radiation treatment, chemotherapytreatment, or a combination) in a lab, thus avoiding use of ill patientsto optimize a treatment method. For example, one can attach a recentlyremoved lung to a bioreactor and treat the lung to ablate tissue.Another example of an in vivo use is for tissue engineering.

The engineered tissues of the present invention have use in vivo. Amongthe various uses, mention can be made of methods of in vivo treatment ofsubjects (used interchangeably herein with “patients”, and meant toencompass both human and animals). In general for certain embodiments,methods of treating subjects comprise implanting an engineered tissueaccording to the invention into or on the surface of a subject, whereimplanting of the tissue results in a detectable change in the subject.The detectable change can be any change that can be detected using thenatural senses or using man-made devices. While any type of treatment isenvisioned by the present invention (e.g., therapeutic treatment of adisease or disorder, cosmetic treatment of skin blemishes, etc.), inmany embodiments, the treatment is a therapeutic treatment of a disease,disorder, or other affliction of a subject. As such, a detectable changemay be detection of a change, preferably an improvement, in at least oneclinical symptom of a disease or disorder affecting the subject.Exemplary in vivo therapeutic methods include regeneration of organsafter treatment for a tumor, preparation of a surgical site forimplantation of a medical device, skin grafting, and replacement of partor all of a tissue or organ, such as one damaged or destroyed by adisease or disorder. Exemplary organs or tissues include: heart, lung,liver, kidney, urinary bladder, brain, ear, eye, or skin. In view of thefact that a subject may be a human or animal, the present invention hasboth medical and veterinary applications.

In one embodiment, the method comprises exposing a tissue to thedecellularization methods of the invention to kill cells of the treatedtissue and to create a tissue scaffold. The method can further compriseseeding the tissue scaffold with cells, and allowing the seeded cells toproliferate in and on the tissue scaffold. Proliferation produces aregenerated tissue that contains healthy and functional cells.

The invention also provides methods of treating a patient by implantingan engineered lung tissue into a mammal in need thereof. In someinstances, the engineered lung tissue comprises suitable cells, forexample NPCs. However, the invention should not be limited to anyparticular type of cells. After implantation, the grafted cells canrespond to environmental cues that will cause it to developcharacteristics of the endogenous tissue. Preferably, the cells formhistiotypic alveolar-like structures, comprised of differentiated distalepithelial cells (proSpC expressing) forming ductal structures. Thus,the implanted cells will develop characteristics that liken it to thesurrounding tissue. Using these methods, the biological scaffolding canaugment the tissue; the biological scaffolding of the invention can beused for tissue engineering and in any conventional tissue engineeringsetting.

Accordingly, the invention encompasses tissue regeneration applications.The objective of the tissue regeneration therapy approach is to deliverhigh densities of repair-competent cells (or cells that can becomecompetent when influenced by the local environment) to the defect sitein a format that optimizes both initial wound mechanics and eventualneotissue production. The composition of the instant invention isparticularly useful in methods to alleviate or treat lung tissue defectsin individuals. Advantageously, the composition of the inventionprovides for improved lung tissue regeneration. Specifically, the tissueregeneration is achieved more rapidly as a result of the inventivecomposition.

Advantageously, the compositions and methods of the invention representan improvement over prior art methods. Preferably the composition foruse in treating a lung tissue defect comprises NPCs, more preferablyNPCs seeded on a scaffold and cultured in vitro to generate a3-dimensional culture, as described elsewhere herein.

Model for Drug Discovery

The present invention provides an in vitro method suitable to allowevaluation of test compounds for therapeutic activity with respect to apulmonary disease or disorder. Preferably, the method includes the useof an engineered three dimensional lung tissue.

The invention is based on a model developed using decellularized tissue.In some instances, the decellularized tissue can be seeded with suitablecells. In some instances, mixed populations of NPC which containepithelial, mesenchymal, and endothelial cells are used to generate thethree dimensional engineered lung tissue. For example, the NPCs areplaced within a three dimensional decellularized lung tissue. Thus, themodel incorporates the influence of NPC on the growth and cell-cellcommunication with neighboring cells. The three dimensional lung tissuemimics a natural lung tissue, for example the engineered lung tissueexhibits branching morphogenesis exemplified by natural lung tissue.

The model is useful for testing drugs on the pathology of a lung tissue.In addition, the model can be used to examine the effects of particulardelivery vehicles for therapeutic agents on the pathology of lungtissue, for example, to compare the effects of the same agentadministered via different delivery systems, or simply to assess whethera delivery vehicle itself (e.g. a viral vector) is capable of affectinglung pathology.

In one embodiment, the invention provides an in vitro method forscreening a test agent for the ability of the test agent to modulate thehealth of a lung tissue. The method comprises contacting a test agent toan engineered three dimensional lung tissue model and measuring theeffect that the test agent has on the lung tissue model. Any alterationto the model in the presence of the test agent is an indication that thetest agent is able to modulate the health of a lung tissue.

In another embodiment, the present invention provides an in vitro methodfor observing an effect a test agent has on a lung tissue, comprisingthe steps of:

a) providing at least one three-dimensional lung tissue model, whereinthe model is intended to model normal lung tissue;b) contacting the test agent with the lung tissue model; andc) observing the effect the test agent has the lung tissue model.

The tissue model is a construct which comprises a three-dimensionalarray of cells on a scaffold, for example a collagen matrix, and atleast one test cell. The method comprises observing the effect of thetest agent on the pathology of the lung tissue. However the method mayfurther comprise the step of observing the effect of the test agent onindividual cell types of the lung tissue.

The test agent may be any agent including chemical agents (such astoxins), pharmaceuticals, peptides, proteins (such as antibodies,cytokines, enzymes, etc.), and nucleic acids, including gene medicinesand introduced genes, which may encode therapeutic agents such asproteins, antisense agents (i.e. nucleic acids comprising a sequencecomplementary to a target RNA expressed in a target cell type, such asRNAi or siRNA), ribozymes, etc. Additionally or alternatively, the testagent may be a physical agent such as radiation (e.g. ionizingradiation, UV-light or heat); these can be tested alone or incombination with chemical and other agents.

The model may also be used to test delivery vehicles. These may be ofany form, from conventional pharmaceutical formulations, to genedelivery vehicles. For example, the model may be used to compare theeffects on a therapeutic effect of the same agent administered by two ormore different delivery systems (e.g. a depot formulation and acontrolled release formulation). It may also be used to investigatewhether a particular vehicle-could have effects of itself on the lungtissue. As the use of gene-based therapeutics increases, the safetyissues associated with the various possible delivery systems becomeincreasingly important. Thus the models of the present invention may beused to investigate the properties of delivery systems for nucleic acidtherapeutics, such as naked DNA or RNA, viral vectors (e.g. retroviralor adenoviral vectors), liposomes, etc. Thus the test agent may be adelivery vehicle of any appropriate type with or without any associatedtherapeutic agent.

The test agent may be added to the model to be tested using any suitablemeans. For example, the test agent may be added drop-wise onto thesurface of the model and allowed to diffuse into or otherwise enter themodel, or it can be added to the nutrient medium and allowed to diffusethrough the collagen gel. The model is also suitable for testing theeffects of physical agents such as ionizing radiation, UV-light or heatalone or in combination with chemical agents (for example, inphotodynamic therapy).

Observing the effect the test agent has on the model can be accomplishedusing a variety of methods. For example, a particular agent may induce acell to enter apoptosis. Detectable changes in the cell may comprisechanges in cell area, volume, shape, morphology, marker expression (e.g.cell surface marker expression) or other suitable characteristic, suchas chromosomal fragmentation. Cell number may also be monitored in orderto observe the effects of a test agent on cell proliferation; this maybe analyzed directly, e.g. by counting the number of a particular celltype present, or indirectly, e.g. by measuring the size of a particularcell mass. These may be observed directly or indirectly on the intactmodel using, for example, suitable fluorescent cell staining. This canbe by pre-labeling of cells with vital dyes or genetically introducedfluorescent markers (for example green fluorescent proteins) for serialanalysis of the living model or by fixation and post-labeling withfluorescent substances such as propidium iodide or fluorescently labeledantibodies. Alternatively, models may be processed by normalhistological methods, such as immunohistochemistry, using antibodiesdirected against a suitable cellular target, or in situ hybridization,to test for expression of a particular mRNA species. Moreover, this maybe carried out in an automated/robotic or semi-automated manner, usingcomputer systems and software to image the cells at various time pointsand detect any change in, for example, cell density, location and/ormorphology. Confocal laser scanning microscopy in particular permitsthree-dimensional analysis of intact models. Thus it is possible toapply directly to the intact, three-dimensional lung tissue model,quantitative analysis of cell behavior which are normally only possiblefor cells in conventional two-dimensional culture. By this meansquantitative, serial analysis of cell proliferation, apoptosis,necrosis, migration and matrix invasion, among others, are obtained in athree-dimensional lung tissue model which bridges the gap betweenconventional two-dimensional cell cultures and live animal models.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1: Decellularization of Rat Lung and MorphologicalCharacterization of Decellularized Scaffolds

A decellularized organ offers several advantages for use as a tissueengineering scaffold. In one aspect, the decellularized scaffoldcontains the appropriate 3-dimensional organization required for tissuefunction, including a vascular system and airway network in the case oflung. In addition, extracellular matrix (ECM) components are widelyconserved across species, thus reducing the likelihood of adecellularized scaffold inducing an immune response upon xenogeneicimplantation [Bernard et al., 1983, Biochemistry 1983; 22:5213-23]. Inanother aspect, native ECM offers the optimal substrate for cellattachment, spreading, growth and differentiation.

The goal of the decellularization process of the present invention is toremove cellular and nuclear material while retaining key aspects of andminimizing any damage to the ECM of the lung. The results presentedherein demonstrate that native lung tissue can be decellularized toremove cellular components and antigenic molecules, yet retain keyextracellular matrix molecules. In one aspect, a goal of thedecellularization process of the invention is to generate adecellularized lung scaffold that is fully compatible with cell cultureand at the same time provide a barrier function. In addition, it isdesirable for the decellularized lung scaffold to have an intact airwaytree and vascular network.

A chemical methodology for decellularization was used in the presentstudy. The chemicals used in this study included sodium chloride, CHAPS,and EDTA. A hypertonic sodium chloride solution can efficiently lysecells, although it does not assist in removing cellular components fromthe tissue. CHAPS is a zwitterionic detergent, which allows efficientsolubilization and thus removal of cellular material. EDTA is achelating agent that binds key divalent ions (i.e. Ca2+) that aids indisrupting cell attachment to the ECM. In addition, the solution is ofhigh alkalinity, which helps solubilize cytoplasmic cellular componentsas well as GAGs which otherwise clog the matrix [Gilbert et al., 2008 JSurg Res 152(1):135-9].

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

Organ Harvest

Lungs were harvested from young adult (3 month-old) male Fischer 344rats. All animal experimental work was performed with approval from theYale University Institutional Animal Care and Use Committee. Animalswere anesthetized via intraperitoneal injection of sodium pentobarbital(Sigma, 40 mg/kg). After induction of anesthesia, the abdomen wasentered via a transverse incision just below the costal margin. Thediaphragm was punctured, and the rib cage was cut to reveal the lungs.The lungs were perfused via the right ventricle with PBS containing 50U/ml heparin (Sigma). After perfusion was complete, the heart, lungs andtrachea were dissected free and removed en bloc.

Bioreactor Components

Bioreactor components were obtained from Cole-Parmer (Vernon Hills,Ill.). A silicone stopper and 500 ml glass jar formed the basis of thebioreactor. PharMed tubing (Westlake, Ohio), sizes L/S 14 and L/S 16,was inserted through the silicone stopper to enable the necessaryconnections to the lung, including a perfusion loop and air ventilation.Pressure was monitored using a TruWave pressure transducer (EdwardsLifesciences, Irvine, Calif.) between the perfusion pump and theconnection to the pulmonary artery. Perfusion was accomplished using aMasterflex L/S variable speed roller pump (Masterflex, Vernon Hills,Ill.).

Decellularization Process

Fluid used for decellularization was 8 mM CHAPS, 1M NaCl, 25 mM EDTA inPBS. All chemicals were obtained from Sigma, and PBS was obtained fromGibco. The bioreactor was filled with decellularization fluid, and thebioreactor was transferred to an incubator kept at 37° C. The perfusionpressure was monitored at the inflow to the pulmonary trunk and keptbelow 30 or 20 mmHg. The decellularization fluid was replaced with freshfluid at the following time points: 30 min, 1 hour, 2 hours, 4 hours, 6hours. For most conditions, decellularization was stopped after 4 or 6hours.

DNA Assay

DNA content of tissues was quantified using the Quant-iT PicoGreen dsDNAassay kit (Invitrogen, Eugene, Oreg.), following manufacturer'sinstructions. Briefly, tissue samples were weighed and lyophilized,diluted in TE buffer and mixed with the Quant-iT PicoGreen reagent.Fluorescence was measured at 535 nm with excitation at 485 nm, and DNAcontent was quantified using a standard curve. At least 4 samples weremeasured for both native and decellularized samples.

Western Blot

Tissues for Western blotting were digested in cold RIPA buffer (BostonBioproducts) with added protease inhibitors (Sigma) and homogenized at15,000 rpm for 30 seconds. After incubation for 1 hour at 4° C.,insoluble particles were removed by centrifugation at 14,000 g for 25min. Protein concentration was quantified via Bradford assay [Bradford,1976, Anal Biochem 72:248-54], then boiled in Laemmli's reducing buffer(Boston Bioproducts) for 25 min at 65° C. Samples were stored at −80° C.until analysis. Samples were run on variable percent polyacrylamidegels, using 25-30 μg of protein. After electrophoresis, protein wastransferred to a nitrocellulose membrane. Membranes were rinsed in TBS,then blocked for 1 hour in 5% non-fat dry milk (NFDM) or 3% bovine serumalbumin in TBS with 0.05% tween-20 (TBS-T). Primary antibodies wereapplied overnight in 2% NFDM or 3% BSA in TBS-T. Secondary antibodieswere from Santa Cruz and were raised in either donkey or goat, and wereapplied for 1 hour at room temperature at a dilution of 1:2000. Proteinwas detected using substrate from Supersignal West Pico, which wasapplied for 5 minutes before film development.

Immunofluorescence

Tissue blocks were fixed for 4 hours in 3.7% formaldehyde (Sigma), thentransferred to 70% ethanol and embedded in paraffin. Thin (5 μm)sections were prepared by the Yale University Histology core facility.Tissue sections were deparaffinized in xylene, rehydrated through anethanol gradient, and rinsed in buffer (PBS+0.2% triton-X) for 15minutes. Antigen retrieval was performed in 0.01M citric acid, pH 6.0,at 70° C. for 20 minutes. After cooling to room temperature, sectionswere rinsed in buffer, then blocked in PBS with 5% bovine serum albumin(BSA) and 0.75% glycine for 1 hour at room temperature. Primaryantibodies were applied at the appropriate concentrations in blockingbuffer overnight at 4° C. Slides were rinsed 3 times in buffer and thensecondary antibodies were applied at 1:500 dilution in blocking bufferfor 1 hour at R.T. Secondary antibodies were AlexFluor 555 donkeyanti-goat or goat anti-rabbit and AlexaFluor 488 chicken anti-rabbit,obtained from Invitrogen. Slides were mounted using DAPI-containingmounting media (Vector Labs), and images acquired using a Zeiss Axiovert200M inverted fluorescent microscope.

Scanning Electron Microscopy

Samples were fixed using 2% glutaraldehyde and 2.5% paraformaldehyde in0.1M cacodylate buffer (EMD Biosciences, Gibbstown, N.J.) for 2 hours atroom temperature, then rinsed in cacodylate buffer, sliced, anddehydrated through an ethanol gradient. Samples were further dehydratedin hexamethyldisilizane for 10 min and dried overnight, then sputtercoated with gold and analyzed using a JOEL JXA-8600 at the YaleUniversity Geology and Geophysics facility.

Transmission Electron Microscopy

Samples were fixed using 4% paraformaldehyde in PBS and then placed in2% glutaraldehyde and 2.5% paraformaldehyde in 0.1M sodium cacodylatebuffered fixative (pH 7.4) for 2 hours at room temperature. The sampleswere rinsed 3 times in 0.1M sodium cacodylate buffer and postfixed in 1%osmium tetroxide for 1 hour, then en bloc stained in 2% uranyl acetatein maleate buffer pH 5.2 for a further hour. Then, the samples wererinsed, dehydrated through a graded ethanol series and infiltrated withepon resin and baked overnight at 60° C. Hardened blocks were cut usinga Leica UltraCut UCT and 60 nm sections were collected on nickel gridsand stained using 2% uranyl acetate and lead citrate. Samples wereviewed on a FEI Tencai Biotwin TEM at 80 kV. Images were taken using aMorada CCD digital camera using iTEM (Olympus) software.

Microsphere Retention

Decellularized or native lungs were attached to cannulae as describedelsewhere herein, and the lung was inflated via the trachea with PBScontaining 5 μm microspheres. The vasculature was then flushed with 3rinses of 10 ml PBS. Microspheres were washed twice in dH₂O to removedebris and lyse any cells that would otherwise affect the native lungreadings. Using a Coulter counter set to measure particles between 4.9μm and 5.1 μm, the microsphere concentration in each sample wasquantified and compared to a baseline reading taken before microsphereinjection.

Micro CT Imaging

Native or decellularized lungs were fixed in 10% neutral bufferedformalin (Sigma) and injected with contrast agent through either theairway or vasculature. Contrast agent was 20% bismuth and 5% gelatin(Sigma) in PBS. After injection of contrast, the lung was cooled in anice bath to polymerize the gelatin.

For the whole lung, the pulmonary vasculature was imaged with a micro-CTimaging system (GE eXplore Locus SP, GE Healthcare), set to a 0.029-mmeffective detector pixel size. The micro-CT was operated at 60 kV peakx-ray tube voltage, 80 mA tube current, 1600 millisecond per frame, 22detector binning model, 720 views, and 0.5o increments per view. For thehigh resolution imaging of one lobe (right superiorlobe), samples werepositioned on a computer-controlled rotation stage and scanned 360around the vertical axis in rotation steps of 0.4°. The tube is operatedat an 80 kV peak and 80 mA. The exposure time for each view wastypically 3000 millisecond, with detector binning model set to 1×1 andresolution of 0.0065 mm. Both acquisitions resulted in a set ofcontiguous axial VFF-formatted images through the lung or one lobe.

With the use of Microview Software (GE Healthcare), the raw data wascorrected and reconstructed with voxels of dimensions 58 μm×58 μm×58 μmto visualize the whole vascular tree in the lung. For the high-qualityof the vascular tree (one lobe), voxels of dimensions was set to 6.5μm×6.5 μm×6.5 μm. This software was also used to reconstruct maximumintensity projection images from the raw data.

Multiplanar reformation, spatial filtering, and volume renderingtechniques allowed for the data set to be viewed in transverse,sagittal, coronal, hybrid planes, and 3D format. Binarized images wereused for object extraction and region-of-interest measurements.Three-dimensional volume images are reconstructed from the angular viewsby using a modified Feldkamp filtered back-projection algorithm.However, with this system, an entire rat lung (field of view,approximately 3.0 cm) may be studied, with images having typical cubicvoxel dimensions as small as 58 μm. The opacity of each voxel isrepresented by a 16-bit gray-scale value.

The results of the experiments are now described.

Decellularization Method

The results presented herein demonstrate a decellularization method thatremoves cellular material from complete lobes of intact rodent lungs. Itwas observed that that decellularization using 1M NaCl, 8 mM CHAPS and25 mM EDTA was optimal to remove cellular material and yet did notappear to remove collagen or elastin fibers (based on histology) ordamage the structural integrity of the matrix (based on mechanicaltesting). In comparison, decellularization with solutions containing SDSwere found to damage the mechanical strength of the matrix. Otherconditions were found to either not efficiently remove cellular materialor cause significant declines in the matrix integrity.

Histological Analysis

Histology was used to characterize many decellularized lung scaffolds.Based on H&E staining and DAPI-staining for nuclei and DNA, thedecellularized lungs did not show a single intact cell. On occasion,unwound DNA or cellular antigen was observed, but intact cells were notobserved. FIGS. 1A-C demonstrates H&E staining of native anddecellularized lung, while FIGS. 2A-B shows DAPI-staining for remnantDNA. Preservation of the pulmonary structure was also observed based onthe fact that alveolar septae appeared intact on standard histologicalsections, as do the larger airways and blood vessels.

DNA Content

The complete removal of cellular material is important for severalreasons. First, if the scaffold is intended to be used for tissueengineering applications, one must be certain that all the cells fromthe scaffold are removed before seeding the scaffold with a new cellsource. In addition to complicating the evaluation of the reseededscaffolds, any remaining cellular material would cause immunecomplications if the engineered tissue is used for in vivo applications[Conconi et al., 2005, Transpl Int 18:727-34; Macchiarini et al, 2008,Lancet 372(9655):2023-30; Alexander et al., 2009, Cell Transplant18:255-9]. As a result, the scaffold of the present invention has beenconfirmed that both MHC Class I and II antigens are not present in thedecellularized scaffolds. Second, in order to evaluate separately thecontributions of the extracellular matrix to lung mechanics, allcellular components should be removed. The two classes of componentsthat can contribute to peripheral lung mechanics are cellular materialand the extracellular matrix. Extracellular matrix can be furtherdivided primarily into collagen, elastin, and proteoglycans [Cavalcanteet al., 2005, J Appl Physiol 98:672-9; Dunsmore et al., 1996, Am JPhysiol 270:L3-27; Ito et al., 2005, J Appl Physiol 98:503-11; Suki etal., 2005, J Appl Physiol 98:1892-9]. By ensuring removal of cellularcomponents from the decellularized scaffolds, mechanical properties ofthe scaffold can be assessed.

In order to document removal of cellular material, a quantitative DNAassay was performed. A drastic reduction in DNA content indecellularized scaffolds compared to native lung was observed (FIG. 1C).Decellularized scaffolds contained approximately 1.2% of the DNA foundin native lung, which corresponded to 1.83±0.29 ng of DNA per mg dryweight. This compares to 38.7±5.8 ng/mg for native lung. While extensiverinsing of the scaffolds can be generally used to minimize remnant DNA,complete removal of all DNA was difficult and small amounts of DNAremained, as demonstrated by DAPI stains showing small clusters ofunwound DNA in FIGS. 2A-B. The drastic reduction in DNA content wasindicative of cellular removal, and together with the histologicalfindings confirmed that all viable cellular material was absent from thescaffolds.

In the decellularized scaffolds, it has been demonstrated that almost99% of DNA has been removed. A small amount of DNA remains in thematrix, but is present as elongated strands of DNA, as shown in FIGS.2A-B. It has been observed that no organization of this remnant DNA innuclear structures, based on DAPI staining.

It has been observed that removal of 98.8% of DNA compared to nativelung, with a remaining DNA concentration of 1.83 ng DNA per mg of tissue(dry weight). This compares favorably to levels of 16.6 ng/mg remnantDNA seen by others for decellularized heart tissue [Ott et al., 2008,Nat Med 14:213-21], especially considering that level is standardized towet weight, not dry weight as in this and other studies [Gilbert et al.,2008 J Surg Res 152(1):135-9]. However, the levels of remnant DNAobserved are higher than those seen for commercially available andlaboratory produced ECM scaffolds used for skin grafts, where mostscaffolds show less than 0.2 ng DNA per mg dry weight, although somescaffolds had as much as 1.13 ng/mg remnant DNA [Gilbert et al., 2008 JSurg Res 152(1):135-9].

Immunogenicity

Immunogenicity of the decellularized scaffolds were characterized bystaining for Major histocompatibility complex (MHC) Class I and IIantigens. MHC class I and II proteins are membrane glycoproteins thatare important in the antigen-specific immune response. MHC Class Iantigen is expressed on all nucleated cells, while MHC Class II antigenis found on specialized cells of the immune system. MHC Class I antigensallow an organism to recognize ‘self’ from ‘non-self’, and are thusimportant to remove from the decellularized scaffold in order to avoidimmune problems upon future implantation of engineered lung tissue intoan animal model. FIG. 3 depicts Western blotting results for MHC Class Iand II antigen as well as β-actin. Complete loss of both MHC Class I andII antigens was observed by immunoblotting, confirming that thedecellularized scaffolds would not be expected to provoke a significantimmune response if used for tissue engineering applications. β-actin wasalso lost, consistent with the absence of cellular material. It isbelieved that the scaffolds are unlikely to provoke an immune responseif implanted into a host.

Extracellular Matrix Characterization

Collagen: Collagen is the most important structural component of thelung, being principally responsible for the overall mechanical strengthof the tissue. Immunofluorescence was used to characterize thedistribution of collagens I and IV in native and decellularized lung, asshown in FIGS. 4A-B. Both collagen I and IV are retained by thedecellularized matrix, with collagen I noted principally around thelarger airways and vasculature, and collagen IV noted throughout theparenchyma. Similar staining patterns were noted for both native anddecellularized lung. The preservation of these collagen subtypes intheir anatomically appropriate locations may enable the selectivedeposition of cell types during the development of engineered lungtissue.

Scanning EM Evaluation of Decellularized Scaffolds

Scanning electron microscopy (SEM) was used to evaluate themicrostructure of the decellularized lung scaffolds. FIGS. 5A-B showssample images, demonstrating cellular removal yet overall maintenance ofalveolar architecture. The alveoli in decellularized lungs appearedslightly deflated, which is an artifact of fixation. Native lung wasfixed by inflating the lung with fixative; the decellularized lung,however, cannot contain the fixative fluid within the alveolarcompartment when pressurized, thus giving the lung a deflatedappearance. However, there is a general similarity in alveolararchitecture with preservation of the alveolar septae. These results,together with findings from histology studies, indicate that the overallpulmonary airway architecture and alveolar structure, including alveolarseptae, were intact in the decellularized scaffolds.

Impact of Perfusion Pressure on Scaffold Ultrastructure

In addition to the scanning EM studies, transmission EM (TEM) was usedto study the capillary-alveolar basement membrane. This is a criticalfeature of the decellularized scaffolds as the presence of an intactcapillary network allows the decellularized scaffold to resistmacromolecular transit into the alveolar spaces and also provides asuitable substrate for the growth of capillary endothelium in engineeredlung tissues.

FIGS. 5A and 5B depict TEM images of native lung and lung that wasdecellularized without control of vascular perfusion pressures. Undersuch conditions, the alveolar basement membrane was at times notidentifiable and no capillaries could be found. Without wishing to bebound by any particular theory, it is believed that damage to thebasement membrane and ultrastructure could be reduced by minimizing theperfusion pressures during the decellularization process and maximallyvasodilating the vasculature before beginning decellularization.Although the decellularization fluid was perfused through thevasculature at sub-physiologic flow rates, vascular perfusion pressurecan become supraphysiologic during decellularization due to massive celllysis and buildup of cellular protein and DNA in the vasculature.Therefore the pulmonary arterial pressure was carefully monitored andthe decellularization bioreactor and perfusion rate was modified inorder to keep this pressure strictly below ˜20-30 mmHg. The vasodilatorsodium nitroprusside was utilized to minimize the initial perfusionpressures.

FIG. 6C shows TEM images of scaffolds decellularized with pressures keptbelow ˜30 mmHg. Under these conditions, an intact, continuous alveolarbasement membrane was observed. Collagen fibers and other matrixcomponents are retained within the alveolar septae. However, we do notnotice the presence of any clear capillary structures, which should bepresent in abundance surrounding the alveoli.

Retention of Capillary Structures in Decellularized Scaffolds

The typical pressure in the pulmonary vascular system of the rodent isless than 15 mmHg [Lee et al., 1999, Cell 99:301-12], significantlylower than the 30 mmHg utilized in the above studies. Despite reducingthe perfusion flow rate and using a vasodilator to lower perfusionpressures, it was difficult to maintain the decellularization perfusionpressure below 30 mmHg. However, it was discovered that a slightmodification in the decellularization protocol enabled perfusion duringdecellularization at pressures less than ˜20 mmHg. Of significance, thisenabled the retention of capillary structures. This modificationencompassed lavaging the airway compartment with decellularization fluidbefore beginning perfusion of the decellularization fluid through thevasculature. The result was the significant lowering of the vascularperfusion pressure, especially at the beginning of the decellularizationprocess. As shown in FIG. 8 , this technique enabled the retention ofcapillary structures in the decellularized scaffolds. It is believedthat the retention of capillaries is a significant development in thecreation of decellularized lung scaffolds.

The scaffold should retain an intact airway tree and vascular network.Using scanning and transmission electron microscopy in addition tomicro-CT imaging, it has been demonstrated that, overall, the scaffoldis remarkably well preserved after the decellularization process.Scanning EM, as well as routine histology, demonstrated that thescaffold was grossly intact without large defects (i.e. alveoli andalveolar septae appear intact). Transmission EM demonstrated that thealveolar basement membrane was well preserved and that at least somecapillaries were retained. Micro-CT imaging demonstrated that thevasculature was intact down to vessels of 100 μm diameter.

Permeability Assessment

In order for a lung to function in vivo, it must possess a continuous,patent and non-leaky vasculature in order to avoid massive blood lossinto the alveolar and interstitial spaces. The ability of decellularizedlung scaffolds to retain 5 μm microspheres in the airway compartment,without allowing transport of these macromolecules into the vasculaturewas evaluated. Five μm particles were use in order to mimic the size ofred blood cells, the principle component of blood, which would need tobe retained in the vasculature. Therefore, the leak of 5 μm particlesout of the airway and into the vasculature was evaluated, with theassumption that there was no significant directionality to the movementof such particles across a decellularized membrane.

The permeability of native lungs, lungs decellularized with uncontrolledperfusion pressures (constant perfusion flow rate), and lungsdecellularized after vasodilation and with controlled perfusionpressures (less than 30 mmHg) was determined. The results are shown inFIG. 8 , and confirmed the TEM findings on a larger scale. It wasobserved that decellularization with high (uncontrolled) perfusionpressure lead to a 39% leak, compared to 5.7% for low-pressuredecellularization and 2.1% for native lung.

Micro-CT Imaging

Micro-CT imaging was used to evaluate the patency of the airway andvascular compartments of decellularized lung scaffolds. This techniqueallows obtaining 3-dimensional images of the lung scaffolds, andfacilitate the identification of the degree of patency of the airway andvascular compartments.

FIGS. 9A-B shows images of the vasculature, with resolution of 58 μm. Atthis resolution, the large vessels are shown to be intact (top panels ofFIGS. 9A-B), and the native and decellularized samples are generallysimilar, shown in the lower and middle panels. Higher resolution images(6.8 μm) of the vasculature are shown in FIGS. 10A-B, where vessels areshown as 3-dimensional projections (maximal intensity projections). Inthese images, slight vascular leak was identified as the haziness shownin some areas of the decellularized scaffold.

A critical feature of the decellularized matrix is the preservation ofthe native 3-dimensional structure. In order to evaluate the extent towhich the structure of the decellularized scaffolds was preserved, acombination of scanning and transmission EM, micro-CT, and a microspherepermeability assay was used. The ultrastructural characteristics ofdecellularized lung was examined using SEM, and demonstrated maintenanceof alveolar architecture and alveolar septae. Transmission EMdemonstrated a completely intact alveolar basement membrane as well ascollagen and elastin fibers. These EM findings are consistent with otherwork in decellularizing lung matrix, where such structures are retained[Lwebuga-Mukasa et al., 1986, Exp Cell Res 162:423-35]. With strictcontrol of vascular perfusion pressure during decellularization, theresults presented herein demonstrate the retention of capillaries.Micro-CT imaging demonstrated retention of the vascular network down tovessels of 100 μm diameter, based on conservative estimates, with asubstantial number of smaller vessels also intact.

Example 2: Contribution of Extracellular Matrix Components to theMechanical Integrity of Decellularized Lung Tissue

The following experiments were designed to evaluate the composition ofthe decellularized scaffolds in more detail with a focus on themechanical properties of the scaffolds. Without wishing to be bound byany particular theory, it is believed that that decellularized lungscaffolds retain salient mechanical features of native lung, dueprincipally to contributions from collagen and elastin. The resultspresented herein demonstrate the utility of the decellularized lungtissue as a platform to study lung mechanics independent of cellularcontributions.

The results presented herein demonstrate that collagen content isretained, elastin content is retained at ˜40% of native levels, whileglycosaminoglycans are largely lost from the decellularized scaffolds.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

Organ Harvest and Decellularization

Lung tissue was harvested and decellularized as described elsewhereherein.

Histological Analysis

Histology was used to characterize many decellularized lung scaffolds,and to confirm the removal of cellular material. Tissues were fixed,paraffin-embedded and sectioned. Analysis was performed with standardhematoxylin and eosin staining (H&E), Masson's trichrome for collagen,Verhoff van Gieson for elastin, and Alcian blue for proteoglycans, aswell as staining for DNA using 4′,6-diamidino-2-phenylindole (DAPI).

Collagen Assay

Collagen was quantified with a colorimetric assay to detect OH-Prolineusing a modified Grants method [Grant, 1964, 1964, J Clin Pathol17:685-6]. Lung samples were lyophilized and weighed, then incubated inpapain (140 μg/ml) at 60° C. overnight (Sigma). Papain-digested sampleswere incubated in 6 N HCl at 115° C. for 18 hours, neutralized, oxidizedwith chloramine-T, and reacted with p-dimethylaminobenzaldehyde.Absorbance was measured at a wavelength of 550 nm and a 1:10 w/w ratioof hydroxyproline to collagen was used to calculate the collagen contentof the tissue. At least 4 samples were measured for native anddecellularized samples.

Elastin Assay

Elastin was quantified using the Fastin Elastin assay kit (Biocolor,Belfast, N. Ireland). Lung samples were first lyophilized and weighed,and then the elastin was extracted following the method described inForonjy et al. [Foronjy et al., 2008, Am J Physiol Lung Cell Mol Physiol294:L1149-57]. Samples were incubated with 0.25M oxalic acid at 100° C.,then centrifuged at 10,000 g and the supernatant saved. The supernatantfrom 5 extractions was pooled, and the supernatant from the 6thextraction was also measured to ensure that no more elastin remained inthe tissue. The oxalic acid was cleared using a 10,000 molecular weightcutoff filter (Millipore), then resuspended in dH2O and analyzed usingthe Fastin Elastin kit according to the manufacturer's instructions. Atleast 4 samples were measured for native and decellularized samples.

Sulfated Glycosaminoglycan Assay

Sulfated glycosaminoglycans (sGAGs), including chondroitin, dermatan,heparan and keratan sulfates, were quantified using the Blyscan GAGassay kit. Papain-digested samples (prepared as described for thecollagen assay, above) were assayed according to the manufacturer'sinstructions. Briefly, sulfated GAGs were labelled with1,9-dimethyl-methylene blue dye and absorbance was measured at 650 nm.

Mechanical Testing

Native and decellularized lung samples were analyzed using an Instron5848 equipped with a 10N load cell. Slices of tissue of known dimensionswere cyclically pre-stretched for 10 cycles to 20% strain to investigateelastic properties and then stretched until failure to evaluate ultimatetensile strength (UTS). See FIG. 11 for a schematic of the testingprotocol. Using tissue dimensions, engineering stress and engineeringstrain were calculated from force and distance.

The results of the experiments are now described.

Collagen and Elastin Content

As shown in FIG. 12C, collagen content in decellularized scaffolds wasindistinguishable from native lung. This preservation of collagen isimportant as collagen plays a key role in the mechanical strength of thelung. Collagen content was also maintained on histochemical staining viaMasson's trichrome, shown in FIGS. 12A-C. Also shown in FIG. 12C,collagen content was decreased in scaffolds decellularized with SDS, oneof the decellularization methods that was not found to be suitable. Itis believed that this loss of collagen correlates with decreasedmechanical integrity in SDS decellularized scaffolds.

Elastin content is also preserved, although diminished, in thedecellularized scaffolds, as demonstrated by both quantitative assay andhistological staining (FIGS. 13A-C). Elastin fibers allow for theelasticity of the lung, critical to the natural recoil of the tissuethat plays a key role in the relaxation and thus exhalation of the lungafter inhalation. The retention of these fibers through thedecellularization process is critical, as it allows the lung scaffold tobe properly ventilated during efforts at reseeding the scaffold withpulmonary cell populations. Although the scaffolds lost 60% of thenative elastin content, the remaining elastin was sufficient to allowelastic function of the lungs, as seen from the mechanical testingresults discussed elsewhere herein.

Overall, the retention of these key ECM components allowed the scaffoldto undergo physiological levels of mechanical stress, which is importantas a variety of developmental and cell differentiation processes rely onmechanical stimuli. In addition, the ECM is critical in aiding cellattachment to the matrix, and the retention of these native ECMcomponents facilitate cell attachment and spreading and thus thedevelopment of bioengineered lung tissues.

Proteoglycan Content

Proteoglycans consist of a core protein linked to one or moreglycosaminoglycan (GAG) chains. Most GAGS are sulfated, enabling theirdetection via quantitative assay, the results of which are shown inFIGS. 14A-C. It was observed that the GAG content of the decellularizedscaffolds was significantly lower than native lung (˜6% of native lunglevels). Proteoglycans are found either on the cell surface or withinthe extracellular matrix [Ferdous et al., 2007, Tissue Engineering13:1893-904], and their removal is due in part to the removal ofcell-bound GAGs. However, the GAGs found within the ECM can also besolubilized via the decellularization solutions. FIGS. 14A-C, is analcian blue histological staining for proteoglycans, which show that theamount of GAGs remaining in the decellularized lung scaffolds wasdiminished compared to native lung, confirming the quantitative assayresults.

Mechanical Characterization

Mechanical testing of peripheral lung strips was used to evaluate thequasistatic mechanics of both native and decellularized lung samples.The elastic regions of the stress-strain curves indicate that bothnative and decellularized samples demonstrated hysteretic behavior.Hysteresis demonstrates that lung is a viscoelastic material, and thedifference between the expanding and relaxing curves represents energythat is not recovered during relaxation. In addition, samples did notcreep, as shown in FIG. 15 . If lung tissue were to creep, it would notdeflate to its original position after an inflation; thus, the lungwould never fully deflate and gas exchange would be impaired. Thispreservation of appropriate elastic lung behavior is important for alung scaffold because loss of pulmonary elasticity is seen in severaldisease states, notably emphysema [Gelb et al., 2002, Chest 121:715-21].

Ultimate tensile strength (UTS) is the stress on a sample at failure,and is a measure of a material's strength. As demonstrated in FIG. 16 ,the UTS of decellularized samples was indistinguishable from that ofnative samples. If samples were decellularized in buffers containingsodium dodecyl sulfate (SDS), however, mechanical integrity wascompromised as demonstrated by the decrease in UTS. SDS can degradecollagen, causing fragmentation and swelling of tissues [Bodnar et al.,1986, Thorac Cardiovasc Surg. 34(2):82-5; Gilbert et al., 2006,Biomaterials 27:3675-83] and has also been shown to increase tissueextensibility [Mirsadraee et al., 2006, Tissue Eng 12:763-73]. SDS is ahighly ionic, amphipathic detergent, and its hydrophobic region caninteract with proteins while the hydrophilic portion, especially whennegatively charged, binds water and causes tissue swelling [Bodnar etal., 1986, Thorac Cardiovasc Surg. 34(2):82-5]. Although other studieshave not always seen a decrease in UTS with SDS treatment [Mirsadraee etal., 2006, Tissue Eng 12:763-73], this may be due to tissue differences.Mirsadraee et al. studied pericardial tissue, which contains much moredensely packed collagen fibers than lung. In lung, due to the geometryof the tissue, collagen fibers are highly distributed, and SDS-inducedswelling can much more easily lead to collagen removal, as seen in thequantitative collagen assay.

The results presented herein demonstrate that the decellularizedscaffolds can withstand relevant in vivo physiological forces.

The results presented herein confirmed that both collagen and elastinwas preserved at functional levels. These findings confirmed that theprincipal contributors to lung mechanics are from collagen and elastin,and not from cellular constituents or proteoglycans. The resultspresented herein demonstrate the production of decellularized lungscaffolds that display characteristics of native lung, which make thempromising substrates for tissue engineering applications as well as aplatform for the study of detailed matrix mechanics and lung biology,development and physiology.

Example 3: Design and Validation of a Bioreactor for the In VitroCulture of 3-Dimensional Lung Tissues

A bioreactor can be used to culture 3-dimensional lung tissue in vitro.The development of such a bioreactor would be beneficial not only toresearch on the growth of engineered lung tissue, but to the study ofpulmonary biology. There are currently no available systems that allowthe long term in vitro culture of adult lung tissue.

The following experiments were conducted for the design of a bioreactorfor the in vitro culturing of whole lung tissue. The bioreactor wasdesigned to meet a series of design constraints aimed at the ability toprovide sufficient nutrient supply and mechanical stimulation to thelung tissue in order to support cell survival and differentiation.Experiments were designed to evaluate whether the bioreactor couldsupport the in vitro culture of whole lobes of lung tissue, demonstratedby maintenance of cell viability and differentiation state. In theprocess of evaluating the bioreactor, the effects of perfusion andventilation on lung survival in the bioreactor was assessed. The resultspresented herein demonstrate that the bioreactor can be used for invitro lung tissue culture and therefore applicable for engineering lungtissue.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

Whole Lung Culture

Lungs were harvested from young adult (3 month-old) male Fischer 344rats. All animal experimental work was approved by the Yale UniversityInstitutional Animal Care and Use Committee. Animals were anesthetizedvia intraperitoneal injection of sodium pentobarbital (Sigma, 40 mg/kg).After induction of anesthesia, the chest and abdomen were sprayed withethanol and a transverse incision made just below the costal margin,entering the abdominal cavity. The diaphragm was punctured, and the ribsretracted, taking care not to touch the lungs. The inferior vena cavawas severed and the lungs perfused via the right ventricle with 20-30 mlof PBS containing 50 U/ml heparin (Sigma) and 1 μg/ml sodiumnitroprusside (Sigma). The trachea was then dissected and cut as high aspossible. All remaining connections to the heart and lungs weredissected free, allowing removal of the heart, lungs and trachea en blocfrom the animal.

Cannula Attachment

After removal of organs, cannulae were connected to the trachea and tothe pulmonary artery trunk via the right side of the heart. The apex ofthe heart was cut off with a scalpel, and a right angle cannula wasinserted through the right ventricle and into the pulmonary trunk. Asyringe was attached to this cannula and 5-10 ml of heparinized salinewas injected to ensure proper cannula placement and adequate perfusionof the lungs without leakage. This cannula was then secured with sutureto the heart. A separate straight, barb-end cannula was inserted intothe trachea and secured with suture. The lungs were then connected tothe bioreactor, and decellularized following the protocol describedelsewhere herein.

Cannulae were attached to the pulmonary artery via the right ventricleand to the trachea, and the lung was connected to the bioreactor. Theairway was lavaged with 2% amphotericin, penicillin and streptomycin inPBS, followed by two lavages with PBS, and the bioreactor was thenfilled with media and culture begun. Vascular perfusion and ventilationwere performed as dictated by the experimental conditions.

Bioreactor Components and Assembly

Bioreactor components were obtained from Cole-Parmer (Vernon Hills,Ill.) unless otherwise noted. A silicone stopper and 500 ml glass jarformed the basis of the bioreactor. PharMed tubing (Westlake, Ohio),sizes L/S 14 and L/S 16, was inserted through the silicone stopper toenable the necessary connections to the lung, including a perfusionloop, tracheal connection, air ventilation, and media exchange ports.Pressure was monitored using a TruWave pressure transducer (EdwardsLifesciences, Irvine, Calif.) between the perfusion pump and theconnection to the pulmonary artery. Perfusion was accomplished using aMasterflex L/S variable speed roller pump (Masterflex, Vernon Hills,Ill.). Ventilation was performed using a multichannel programmablesyringe pump (Cole Parmer), with inhalation and exhalation eachperformed over 30 seconds using a volume of 10 ml. A diagram of thebioreactor is shown in FIGS. 17A-C.

Histology and Immunofluorescence

After the desired culture period, lungs were fixed, paraffin-embeddedand sectioned. Routine histology (H&E) was performed, as well asimmunofluorescence for aquaporin-5 (type I epithelium), surfactantprotein C (type II epithelium), CCSP (Clara cells), and PECAM-1(endothelium). Sections were deparaffinized in xylene, rehydrated, andincubated with PBS with 0.2% triton-X (buffer) for 15 minutes. Antigenretrieval was performed using 0.02M citric acid in PBS for 20 min at75-85° C., after which sections were rinsed in buffer. Blocking wasperformed for 1 hour at room temperature with PBS+1% bovine serumalbumin and 0.75% glycine. Primary antibodies were rinsed off withbuffer and secondary antibody was applied for 1 hour at room temperatureat 1:500 dilution. Secondary antibodies were obtained from Invitrogen(AlexaFluor 555 or AlexaFluor 488x). Images were acquired with a ZeissAxiovert 200M inverted fluorescent microscope.

Cell proliferation was assessed via staining for proliferating cellnuclear antigen (PCNA) (Zymed, San Francisco, Calif.), and apoptoticnuclei were detected with terminal deoxynucleotidyl transferase dUTPnick end labeling (TUNEL) stain (Calbiochem, San Diego, Calif.).Manufacturer's instructions were followed for both assays.

Microsphere Ventilation Assay

In order to determine if ventilation of lungs in the bioreactor wassufficient to induce movement of media to perfuse the vasculature, asimple assay was developed using 5 μm polystyrene microspheres (SPISupplies, West Chester Pa.). Lungs were connected to the bioreactor, asdescribed elsewhere herein, and ventilated but not perfused. Thebioreactor chamber was filled with 100 ml of media containing 10 millionmicrospheres (0.1 million microspheres per ml of media). The culture wasallowed to proceed, with ventilation only, for 3 hrs. The lung was thenfixed, paraffin-embedded, sectioned and analyzed using routine histology(H&E).

The results of the experiments are now described.

Bioreactor Design Requirements

The bioreactor incorporates key features of the rodent in vivoenvironment but was also designed allow the user to modify several keyparameters depending upon the desired conditions. The design goals areas follows:

-   -   System must be capable of perfusing media through the        vasculature at a rate specified by the user and within the        physiological levels.    -   System must be capable of ventilating the lungs with air or        media through the trachea. Negative pressure ventilation and the        ability to constantly ventilate the lungs is preferable, in        order to be consistent with normal physiological conditions.    -   Bioreactor should preferably allow for different media types to        bathe the vascular and airway compartments of the lung.    -   Bioreactor must allow for gas exchange into the culture medium,        while simultaneously meeting the above requirements for        ventilation.    -   Bioreactor must have ports to allow for pressure measurements of        the pulmonary artery and tracheal pressures. Pressures should        ideally be within normal physiological values, with a pulmonary        artery pressure of less than 15-30 mmHg [Li et al., 2004, Proc        Natl Acad Sci USA 101:11488-93].    -   Bioreactor must have a means of allowing media exchange on a        periodic basis.    -   Bioreactor must be small and self-contained such that it can fit        within the physical confines of a standard tissue culture        incubator.    -   All bioreactor components must be inexpensive and easily        available.    -   Bioreactor and all components must be able to be sterilized,        preferably via autoclave.        A bioreactor was designed and built that met the above criteria.        A schematic of the bioreactor is shown in FIGS. 17A-C.

Bioreactor Perfusion System

Perfusion to the lung was provided via a roller pump that circulatesmedia from the main bioreactor into the pulmonary artery. The perfusionrate can be specified by the user. The heart of the rat is kept attachedto the lung in order to facilitate the connection of a cannula to thepulmonary arterial trunk through the right ventricle of the heart.However, the pulmonary vein was not connected directly to the perfusionloop. Rather, the pulmonary veins drained through the left side of theheart directly into the main bioreactor reservoir. The venous drainageof the lung exits directly into the main bioreactor.

The perfusion rate through the lungs can be set to a user'sspecifications. Physiologic flow rates in the adult rat are 40-80ml/min, although for engineered tissue culture the flow rate istypically much less than this value. In an adult rat, the entire bloodvolume must pass through the lungs in order to become oxygenated,whereas during engineered tissue culture, only sufficient media tosupport the growth of the pulmonary cells must perfused. Thus, perfusionrates during engineered culture are closer to that in a fetal rat, wherethe blood flow to the lung is only 8-10% of the cardiac output due to anormal physiologic shunt [Hislop et al., 2000, Ped Resp Rev 1:321-7].The pressure profile can be controlled to a limited degree usingvasodilators such as sodium nitroprusside, which can be used to reducepulmonary vascular pressure. Typically the perfusion pressure is keptbelow −30 mmHg, the maximum value typically seen in the pulmonaryarterial system [Li et al., 2004, Proc Natl Acad Sci USA 101:11488-93].

Bioreactor Ventilation System

The bioreactor was capable of both positive and negative pressureventilation. In vivo, breathing is normally accomplished via negativepressure ventilation. The diaphragm contracts and the rib cage expandsto create a negative pressure within the thoracic cavity, causing air toflow into the lung to relieve this pressure imbalance. After inhalation,the breathing muscles relax and the lung passively deflates.

Negative pressure ventilation is the primary mode of ventilation in thebioreactor. In order to effect a negative pressure surrounding thelungs, the main chamber of the bioreactor must be completely airtight.This is accomplished by closing off all air and pressure-monitoringvents. Then, a syringe pump is used to withdraw a set volume of air fromthe main bioreactor, creating a negative pressure. The only pathway forthis pressure to be relieved is for media (or air) to flow into thelungs via the trachea, which is connected to a separate reservoir. Thesyringe pump then reverses direction to push air back into the mainbioreactor. This reverses the buildup of negative pressure inside thechamber, and media (or air) flows back into the tracheal reservoir. Thelung deflates passively during this time.

Tracheal Cannula Utilizes One-Way Valve:

As depicted in FIGS. 17A-C, the connection to the trachea involves aY-connector and a one-way valve open to the main bioreactor. This typeof connection is necessary due to leakage of fluid out of the airwaycompartment. During inhalation, a volume of media enters the lung.However, some of this media leaks across the alveolar membrane into theinterstitial space or vasculature. Therefore, not all of the media thatentered the lung during inhalation can be returned to the trachealreservoir during exhalation. The design shown in FIGS. 17A-C,incorporates the feature of allowing all the media to enter the lungduring inhalation. However, during exhalation, media can return to thetracheal reservoir either from the lung or from the main bioreactor viathe one-way valve.

The bioreactor can also utilize positive pressure ventilation, byconnecting the syringe pump directly to the tracheal cannula or trachealreservoir.

Tracheal Inlet Modification:

In the context of the bioreactor, it was observed that duringventilation, the lung airway compartment was not supplied withsufficient fresh media. Without wishing to be bound by any particulartheory, it is believed that this was because largely the same media wasbeing ventilated in and out of the trachea due to the volume of mediacontained in the tubing between the trachea and the separate trachealreservoir, with insufficient fresh media entering the trachea. The “deadspace” in the airway medium flow loop prevented fresh medium fromreaching the lung tissue during breathing. As a result, the bioreactorwas modified such that the media followed a different path into and outof the lung during ventilation, as outlined in FIGS. 17A-C. Due to thismodification, most of the media entering the trachea with each breathwas sourced directly from the tracheal reservoir (and thus ‘fresh’compared to the media that is exiting the trachea).

Oxygen Supply During Bioreactor Culture:

The oxygen content of tissue culture medium in the bioreactor duringlung cultures was measured, in order to ensure that there was sufficientoxygen content. In particular, it is necessary to ensure that there issufficient oxygen delivery during negative pressure ventilation, duringwhich the main bioreactor is air-tight and the only portal for oxygenentry is via the tracheal reservoir. It was found that the oxygentension does not drop significantly over the course of culture, andremained at 6.0-7.0 mg/L, which is the same as the level in normaltissue culture media. These levels exceed normal physiological levels of80-100 mmHg (6-7 mg/L corresponds to a partial pressure of 137-159mmHg).

Bioreactor Pressure Profiles

The pressure profiles in the trachea and pulmonary artery of engineeredlung tissue cultured in the bioreactor was measured, in order to ensurepressures are within expected or physiological limits. FIGS. 18A-B showsrepresentative profiles. The perfusion pressure was typically keptbetween ˜2 and 30 mmHg. In the example given, the baseline perfusionpressure varied between 10-17 mmHg. However, the effects of the negativepressure ventilation were superimposed on this profile, thus loweringthe perfusion pressure to 0-7 mmHg during a negative pressure ‘breath’.This effect is seen physiologically, wherein the pressure drops in thepulmonary vasculature with inspiration. In the bioreactor, the pulmonaryvein drained directly into the main chamber, which also served as the‘thorax’, which is where negative pressure was created in order toventilate the lung. This served to increase transmission of negativepressures from the bioreactor to the perfused vasculature.

From the perfusion pressure profile, the maximum negative ‘thoracic’pressure was ˜−12 mmHg, approximately consistent with physiologicalvalues. Therefore, during an inhalation, this negative pressure wasexerted on the airways, driving fluid (or air) into the lungs from thetracheal reservoir. This pressure gradually decreases up the airwaytree, and was −3 mmHg at the tracheal inlet. Of note, the pressure atthe inlet to the trachea was essentially zero physiologically, as it istied to atmospheric pressure. However, in the bioreactor, this pressureremained slightly negative during inhalation due to the length of tubingbetween the trachea and the tracheal reservoir, where the pressurereaches zero.

Media and Oxygen Requirements

The following results show a series of calculations intended to helpdetermine the volume of media and air required for a rodent lungcultured in the bioreactor.

Tissue Culture Comparison:

During in vitro tissue culture, it is common to feed 5 million cellswith 12 ml medium every 3 days. If it is assumed that the adult rodentlung contains 100 million cells, which corresponds to a mediarequirement of at most 240 ml every 3 days. However, this would be anoverestimate as cells in tissue culture are generally activelyreplicating, while many cells in an intact rodent lung are quiescent andthus have lower media requirements.

Glucose Consumption Requirements:

It has been demonstrated that the glucose consumption of a perfused ratlung is 43 μmol per gram dry weight per hour [Kerr et al., 1979, Am JPhysiol 236:E229-33]. The lung of an adult rat has a dry weight of˜150-250 mg [; Inokawa et al., 2006, Ann Thorac Surg 82:1219-25] whiletissue culture medium typically contains 5.5 mmol/L glucose. Therefore,the lung of an adult rat would require 28-47 ml of tissue culture mediaper day in order to supply its glucose consumption requirements.

Oxygen Requirements:

Pulmonary artery endothelial cells consume 6 nmol of oxygen per millioncells per minute [Xu et al., 2007, Proc Natl Acad Sci USA 104:1342-7],while rat type II epithelial cells consume 1.25 nmol per minute [Dobbset al., 1980, Biochim Biophys Acta 618:510-23]. Assuming 100 millioncells in an adult rat lung and assuming all cells in the lung consumeoxygen at the higher rate, a rat lung would require at most 26 mg ofoxygen per day. Tissue culture media contains approximately 6 mg ofoxygen per liter, and the bioreactor contains approximately 300 ml ofmedia. Thus, the media can provide 1.8 mg of oxygen with each exchangeof fresh media (every 3 days). In addition, oxygen is contained in theair in the bioreactor; there is approximately 200 ml of air in the mainbioreactor. Air in the incubator contains ˜20% O₂, which at sea leveland 37° C., corresponds to ˜260 mg of oxygen per liter of air. Thereforethe air in the bioreactor contains ˜52 mg of oxygen.

The bioreactor of the invention can provide the media and oxygenrequirements of a cultured rodent lung based on the above calculations.Routinely, a total of 240 ml of medium can be supplied in the bioreactor(180 ml in the main bioreactor and 60 ml in the tracheal reservoir), andthe air in the bioreactor can be exchanged daily. These conditions arebelieved to be sufficient to provide more than enough nutrients andoxygen to a cultured lung.

Whole Lung Culture

In order to validate and optimize the design of the lung bioreactor, invitro culture of whole native rodent lungs was used. Lungs were culturedfor up to 7 days in the bioreactor. It has been demonstrated that thebioreactor provided sufficient nutrient supply and mechanicalstimulation to maintain cell survival and differentiation as well aslung morphology.

The culture of native lung was also used in the bioreactor to examinethe effects of bioreactor conditions on cell survival, lung morphology,and maintenance of cellular differentiation state. The effects of airversus liquid (media) ventilation on lung morphology was initiallycompared. The effects of ventilation technique and nutrient delivery oncell survival was then evaluated. The effect of vascular perfusionpressure on cell survival and differentiation was also evaluated. Theability of the bioreactor to maintain cellular differentiation during7-day cultures was also evaluated.

Effects of Ventilation with Air Versus Media on Overall Lung Morphology:

The effects of ventilating lungs cultured in the bioreactor with eithermedia or room air (˜20% O₂) was evaluated. It is believed thatventilation with media would offer improved cell survival as this wouldprovide improved nutrient delivery, which may be more important in thebioreactor as there is no perfused bronchial circulation to supply thelarge airways. However, ventilation with air is the condition to whichadult lungs are conditioned, and pulmonary epithelium is frequentlycultured in the presence of an air-liquid interface, which has beenshown to enable appropriate pulmonary development in fetal rat lungs[Funkhouser et al., 1976, Biochem Biophys Res Comm 70:630-7]. Therefore,experiments were also designed to examine whether ventilation with mediawould result in loss of epithelial differentiation state, due to thelack of an air-liquid interface.

After 3 days of culture, significant differences were noted betweenlungs ventilated with media versus air. As shown in FIGS. 19A-C mediaventilation appeared similar to native lung; however, air-ventilatedlungs showed greatly dilated airways, with cell debris evident in theairway (FIG. 19C). Furthermore, the center panel of FIG. 19C) shows thatthe bronchial and bronchiolar epithelium of air-ventilated lung wascompletely absent, a finding that was consistent across the entire lung.In addition, dilated peripheral airspaces were evident, as shown in theright panel of FIG. 19C.

It was also observed that the airway epithelium was also denuded ifmedia perfused through the vasculature (in addition to ventilation withair), while if media perfused through the vasculature with noventilation, the airway epithelium was intact. This suggests that theloss of airway epithelium is not due to a lack of sufficient media, butis related to effects of air ventilation. It was observed that bronchialcirculation was not perfused for any cultures.

Epithelial cells are often cultured at an air-liquid interface,consistent with their physiologic locations. An air-liquid interface(ALI) is often utilized to induce epithelial differentiation [Gruenertet al., 1995, Am J Physiol 268:L347-60; Wong et al., 2009, J Clin Invest119:336-48; Hosokawa et al., 2007, Connect Tissue Res 48:9-18], and alack of an air-liquid interface can lead to reduce ciliogenesis[Ostrowski et al., 1995, Exp Lung Res 21:957-70; Yeh et al., 2007,Laryngoscope 117:1439-44]. In addition, an air-liquid interface enablesthe maintained secretion of surfactant by type II epithelium whencultured in vitro [Mason et al., 2002, Am J Physiol Lung Cell MolPhysiol 282:L249-58]. Therefore, it was expected that differences incellular differentiation state in the absence of an ALI would occur.However, significant changes in the expression patterns of Clara cellsecretory protein (CCSP), surfactant protein C (SPC), or aquaporin (AQP)in lungs ventilated with media, as shown for cultures performed out to 7days in FIG. 25B was not observed.

Effect of Perfusion on Cell Survival:

The effect of vascular perfusion on cell survival and cellulardifferentiation in cultured native lungs in the bioreactor was examined,with the aim of determining if perfusion alone could support in vitrolung culture, and if so what perfusion pressure was optimal.Complicating these experiments was the fact that, after explantation ofa lung, vascular permeability was rapidly increased. Isolated lungperfusion using pressures of 10 mmHg can cause pulmonary edema within 10minutes [Wierup et al., 2005, J Heart Lung Transplant 24:379-85].Vascular leak was observed within 5-10 minutes of explantation, with3-4% of small particles (28 nm radius) leaking across thealveolar-capillary membrane. Extensive pulmonary microvascular leakcould result in less or even no media delivery to the distal capillariesand venous structures. Therefore, higher vascular perfusion pressuresthan the physiological levels of ˜1-10 mmHg may be required in order todeliver flow distally and keep distal capillaries patent [Li et al.,2004, Proc Natl Acad Sci USA 101:11488-93].

The effect of vascular perfusion pressures on cell survival during 3 daynative lung culture in the bioreactor was examined. As demonstrated inFIGS. 20A-B, higher perfusion pressures of up to 30 mmHg resulted infewer apoptotic cells as well as higher cell density, compared topressures of 10 or 20 mmHg. However, regardless of perfusion pressure,maintenance of cellular differentiation was poor with vascularperfusion. Substantially lower CCSP and SPC levels were observed (FIGS.21A-D), while aquaporin expression was almost completely absent. PECAMexpression was observed in the larger vessels of the vasculature, butdecreased expression was observed in capillaries, as shown in FIGS.22A-B. These experiments demonstrated that perfusion alone was notsufficient to maintain sufficient cell survival or cellulardifferentiation.

Effect of Media Flow Path in the Airway Compartment on Cell Survival:

While ventilation with media permitted the maintenance of lungmorphology and cell differentiation, significantly higher rates ofapoptotic cells in ventilated cultured lungs compared to native wasobserved (see FIGS. 23A-B and 28A-F). It is believe that this was due toinsufficient fresh media delivery, and thus experiments were designed tomodify the bioreactor in order to increase the delivery of fresh mediato the airway compartment during ventilation. As shown in FIGS. 17A-Cand described elsewhere herein, there is a single line connecting themain bioreactor to the tracheal reservoir. This length of tubing isapproximately 40-45 cm and contains 3-3.5 ml of media. Duringventilation, ˜2.5-3.0 ml of media is drawn into the lung during anegative pressure inhalation, and the same volume of media is returnedvia the tubing to the tracheal jar. Therefore, of the ˜2.5-3.0 ml ofmedia that enters the lung during each ‘breath’, this media is not freshbut simply returns into the lung from the tubing. Therefore, the actualmedia delivery to the lung is far less than would be delivered byventilation with fresh media.

The bioreactor design was modified to add a second connection betweenthe lung in the main bioreactor and the tracheal reservoir. Usingone-way check valves, one connection was used for media delivery duringinhalation and the other connection was used for media return duringexhalation. This modification reduced the ‘recycled’ media from ˜2.5-3.0ml to only ˜0.75 ml with each ventilation cycle, and therefore greatlyincreased the delivery of fresh media.

The effects of this bioreactor modification are shown in FIGS. 24A-C,where the additional breathing line was shown to improve cell survival.The percent of apoptotic cells was reduced to 3.9% for ‘loop’ventilation from 21.5% for ventilation with a single line (‘vent only’on FIGS. 27A-B). This compares to 0.5% for native lung.

While ‘loop’ ventilation increases the delivery of media to the lung byreducing the amount of ‘recycled’ medium, media delivery can also beincreased with the addition of vascular perfusion. Perfusion togetherwith ventilation reduced cell apoptosis to 7.9% from 21.5% forsingle-line ventilation alone (FIGS. 23A-B). The ‘loop’ ventilationmodification slightly increased overall cell number compared tosingle-line ventilation (FIG. 23B), but this was not a significantdifference. For both single-line and ‘loop’ ventilation, cell number wasreduced compared to native but not statistically significant.

The results presented herein demonstrate that ventilation alone enablesthe survival of native lung tissue in the bioreactor for during 3-daycultures, provided sufficient fresh media is delivered to the lung usingeither the ‘loop’ ventilation modification or supplemental vascularperfusion. Loop ventilation demonstrates the best overall results,minimizing cellular apoptosis while maximizing cell number in culturedlung tissues.

Cellular Morphology, Cellular Differentiation, and Alveolar Structure:

In order to more fully validate the bioreactor design, 7 day cultures ofnative lung were performed. These cultures utilized ventilation withmedia with the ‘loop’ modification described elsewhere herein, butwithout any vascular perfusion. Vascular perfusion was not utilized,although future studies could explore the addition of perfusion tolong-term ventilated cultures using ‘loop’ ventilation.

Lungs were evaluated via histology for cell proliferation, apoptosis,and maintenance of cellular differentiation via staining for aquaporin-5(type I epithelium), surfactant protein C (type II epithelium), Claracell secretory protein (Clara cells), and PECAM-1 (endothelium). Overallpulmonary architecture was preserved, including alveolar structure, asshown in FIGS. 25A and 25B. Lower magnification images were notdistinguishable from those shown in FIGS. 19A-C for media breathing. Inaddition, as shown in FIGS. 25C through 25J, patterns of expression ofcellular markers were not substantially different from native lung.

Ventilation Alone Enables Passive Perfusion of the Vasculature of theLung:

It has been demonstrated that ventilation alone can enable cell survivaland maintenance of cellular phenotype of several key lung cell types,including endothelium, for up to 7 days. However, this is in the absenceof active perfusion of medium through the vasculature, which wasinitially surprising. In order to investigate why the lack of perfusiondid not affect endothelial survival or differentiation, an experimentusing 5 μm microspheres to investigate the effect of ventilation on themovement of fluid into the vasculature was performed. It is believedthat the physical movements induced by ventilation were sufficient tocause passive movement of media into and out of the vasculature, whichis open to the media in the bioreactor. In this experiment, lungs wereventilated for 3 hours in the bioreactor which was filled with mediacontaining 5 μm microspheres. If microspheres were observed in thevasculature of the lung, this indicates that passive perfusion wasinduced by ventilation. As demonstrated in FIG. 26 , microspheres werefound in both large vessels as well as some capillaries. These resultsindicate that ventilation alone is sufficient to induce media movementin the vasculature, thus allowing maintenance of the endothelium despitethe lack of perfusion.

This movement of media in the vasculature is a result of the physicalmotion of the lung due to ventilation. Diffusion alone would beinsufficient to move such large particles into the vasculature. Theexpected microsphere movement into the vasculature due to diffusionusing Fick's second law can be approximated.

Supporting Cell Survival and Differentiation

The results presented herein demonstrated that vascular perfusion alonewas not sufficient to support cell survival and cell differentiation,including surfactant production by type II epithelium. However, negativepressure ventilation with media was sufficient to support extensive cellsurvival (to 95.1% of native levels) as well as maintain thedifferentiation of epithelium and endothelium.

The overall objective of the experiments of the Example was todemonstrate the valid design of a bioreactor capable of culturing wholerodent lungs in vitro for long time periods, with the objective of usingthis bioreactor for the future culture of engineered lung tissues. Theresults demonstrate a successful design that is suitable for use inengineered lung culture.

Example 4: Epithelial Development in Engineered Lung Tissues

The results presented herein demonstrate that the decellularizedscaffolds are not cytotoxic and support the adherence and proliferationof a wide range of pulmonary cell types, including epithelial,endothelial, and mesenchymal cells. In some instances, stem cells can beused to engineer a lung tissue. In order for engineered lung tissue tobe useful, it must be able to connect to a vasculature and an airway.The airway must be continuous with alveoli, while the vascularconnections must lead to a dense capillary network surrounding thealveoli.

The results presented herein demonstrate that the development ofengineered lung tissues is affected by key bioreactor conditions,including medium type, perfusion, ventilation, and the presence of anair-liquid interface. In some instances, ventilation with culture medium(i.e. “liquid ventilation”) aids in the differentiation of airwaystructures and epithelial cells. In addition, static culture of smallpieces of engineered tissue at an air-liquid interface exhibitedsignificant effects on tissue growth, and that in the bioreactor,ventilation with air affects cellular differentiation and thedevelopment of epithelial structures.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

Scaffold Preparation

The lungs of adult Fischer 344 rats were harvested and decellularized asdescribed elsewhere herein. Following the decellularization procedure,the scaffolds were rinsed in 10 changes of sterile water, followed byrinsing for at least 12 hours in 10% penicillin/streptomycin in PBS. Inlater cultures, lungs were also rinsed in 10% FBS to aid removal of DNAremnants. The lungs were then transferred to a new, sterile bioreactorwith a complete perfusion and breathing system attached. The lungs weresubsequently rinsed twice in PBS and once in the media to be used forculture.

Neonatal Cell Isolation

Lungs were isolated from neonatal (˜7 day-old) rats as discussedelsewhere herein. Lungs were then rinsed for 10 sec in 70% ethanol andrinsed twice in Dulbecco's modified Eagle's medium (DMEM, Gibco), andthen transferred to a sterile, dry Petri dish. Lungs were minced for 5minutes with a scalpel, and then transferred to a conical tube forelastase digestion. DNase, collagenase and elastase were obtained fromWorthington Biochemical (Lakewood, N.J.). Elastase digestion wasperformed for 20 minutes at room temperature with agitation, using 4U/ml elastase in DMEM with 100 U/ml DNase. Tissue chunks weresubsequently filtered through a 70 μm nylon filter and rinsed with DMEM.Undigested chunks were transferred to a clean tube and digested withcollagenase for 1 hour at room temperature with agitation, in a solutionof 1 mg/ml collagenase in 1:1 DMEM:PBS with Ca2+ and Mg2+.Collagenase-digested tissue was again filtered through a 70 μm filterand undigested pieces were physically crushed using a syringe plunger.The remaining tissue was rinsed with DMEM and filtered through a 70 μmfilter. Cells from the collagenase and elastase digestions werecombined, then washed three times in DMEM and once in the media to beused for culture. Cell viability was assessed using trypan blue dyeexclusion and cells were then seeded into the decellularized scaffoldsas described elsewhere herein.

Neonatal Cell Seeding

After pulmonary cell isolation and preparation of the decellularizedscaffold, the isolated cells were suspended in the medium to be used forculture. For seeding of the airway compartment, 15 ml of cell suspensionper bioreactor was injected into the tracheal reservoir and cells wereseeded by negative pressure ventilation to transfer the cells into theairway compartment of the lung. For seeding of the vasculature, 3 ml ofcell suspension per bioreactor was injected into the pulmonary artery.The cells were allowed to adhere overnight without perfusion orventilation, after which perfusion and/or ventilation was begundepending on experimental conditions.

Engineered Tissue Culture

After seeding, the lungs were cultured statically overnight, after whichperfusion or ventilation was begun. Perfusion and ventilation werevaried according to experimental conditions. Culture medium was replacedtwice weekly. For ventilation conditions, lungs were ventilatedcontinuously except for a brief pause daily in order to allow manualexchange of air in the bioreactor. For the culture of pieces ofengineered tissue, after overnight seeding, scaffolds were removed fromthe bioreactor and cut into small (1-3 mm) pieces using sterilescissors. The pieces were transferred to Petri dishes for culture and,if indicated, later transferred to a Petri dish with a 0.4 μm filterinsert for air-liquid interface culture.

Flow Cytometry

After cell isolation, cells were rinsed in buffer (PBS with 2 mM EDTAand 0.5% bovine serum albumin). For staining of intracellular antigens,cells were fixed with 1% formaldehyde for 15 minutes at roomtemperature, then permeabilized with 0.2% triton-X in PBS. Primaryantibodies were applied in buffer for 30 minutes at R.T. at 1:100dilution. After 3 rinses in buffer, secondary antibodies were appliedfor 20 minutes at room temperature at 1:100 dilution. Cells wereanalyzed on Becton-Dickinson FACSCalibur machines at the Yale School ofMedicine Cell Sorting Facility.

The results of the experiments are now described.

Scaffold is not Cytotoxic

Initially, MLE-12, a tumor-derived lung epithelial cell line was usedfor preliminary culture experiments on the decellularized lungscaffolds. These experiments were performed to demonstrate that thescaffold was not cytotoxic, as well as to demonstrate the first-ordervalidity of the bioreactor system for cultures utilizing decellularizedscaffolds. It was observed that the MLE-12 cell line exhibited robustcell growth during culture periods of up to 10 days on decellularizedscaffolds in the bioreactor, with perfusion of media through thevasculature. Histology is demonstrated in FIGS. 27A-B. Cells appeared toform very primitive alveolar structures at 3 days, but subsequentlyproliferated extensively, and uncontrolled cell growth is shown by 7days, an expected outcome as this is a tumor-derived cell line. Theseexperiments were a preliminary step in the validation of the bioreactorand the decellularized lung scaffolds, and justified the subsequentexperiments using freshly isolated neonatal pulmonary cells.

Harvest of Neonatal Pulmonary Cells

Neonatal pulmonary cells were chosen for several reasons, including theability to isolate a large number of cells which represent aheterogeneous mix of pulmonary cell types, because rodent lungepithelium is difficult to culture in vitro and because the pulmonarycells of neonatal rats are young and relatively plastic [Massaro et al.,1985, J Clin Invest 76:1297-305; Meyrick et al., 1982, Am Rev Respir Dis125:468-73].

Conditions for cell isolation were optimized based on cell number,viability, and distribution of cell types based on flow cytometry.Primary markers used were surfactant protein C (SPC; type IIpneumocytes), aquaporin-5 (AQP; type I pneumocytes), Clara cellsecretory protein (CCSP; Clara cells), and platelet endothelial celladhesion molecule-1 (PECAM-1; endothelial cells). Conditions for cellisolation were chosen as a result of iterated experiments that optimizedoverall cell number and viability. The selection of enzymes andincubation conditions was optimized based on cell yield and viability,as assessed by total cell number, trypan blue dye exclusion, and flowcytometry analysis.

Flow cytometry data of a sample lung isolation that was obtained underan ‘optimized’ isolation regimen is shown in FIGS. 28A-F. In a typicalisolation, 5-10% of cells are CCSP-positive, 40-60% of cells areSPC-positive, 2-8% of cells are AQP-positive, 1-2% of cells arecytokeratin-14-positive, 10-30% of cells are PECAM-1 positive, and 5-10%of cells are α-actin-positive. Using cytospin preparations, staining forCCSP and SPC was confirmed. While most of these percentages are withinexpected range, one would expect higher yield of type I pneumoytes(AQP-positive), based solely on its prevalence in native lung. However,type I pneumocytes are very fragile and many of them are unlikely tosurvive the cell harvesting process. Total cell yield from a litter ofpups (7-12 pups) was approximately 100 million cells, with viability of75-85%.

Preliminary Identification of Bioreactor Conditions for Engineered LungCulture

Experiments were designed to explore various variables and suitableconditions based primarily on cell density, viability and morphology viahistology, as well as some evaluation of protein expression. Theconditions that were evaluated are briefly addressed below.

Medium choice: Several medium compositions, varying both the base mediumand serum concentration were evaluated. Epithelial repopulation,principally type II epithelium, using several medium conditions,including BGJb (serum-free), DMEM with 10% FBS, EGM-2 with 15% FBS, anda 3 part to 1 mix of EGM-2+15% FBS and BGJb was observed. The mediumtypes BGJb and DMEM+10% FBS provided superior conditions for overallepithelial growth, based on histology and immunofluorescence staining.As a result, these media were used for subsequent experiments. However,the invention should not be limited to any specific medium. This isbecause any medium that promotes the desired proliferation anddifferentiation can be used.

Perfusion and ventilation: Both perfusion and ventilation was usedduring the preliminary experiments. For example, cultures were perfusedat 2-5 ml/min in order to provide a nutrient supply. The effect ofventilation once-daily with a single breath was also evaluated. However,significant differences in the resulting engineered lung cultures wasnot detected. Despite not observing a clear benefit from the once-dailyventilation, it was decided that this minimal level of ventilation wouldprovide a more physiological culture environment. Therefore, for themajority of subsequent experiments, the cultures were perfused andventilated once daily with a single breath.

Decellularized Scaffolds Support the Growth of Epithelial, Endothelialand Mesenchymal Cells

The following experiments were designed to demonstrate the validity ofthe decellularized scaffolds, the lung bioreactor, and the isolatedneonatal pulmonary cell population for the development engineered lungtissue. The precise conditions used in the culture of these engineeredtissues are identified in Table 1. Also described in the table areconditions that were specifically probed to evaluate the effects ofthose conditions on engineered tissue growth. However, the invention isnot limited to these conditions. Rather, any applicable condition isencompassed in the invention so long as the conditions promotegeneration of an engineered lung in the context of the bioreactor.

TABLE 1 Bioreactor conditions for engineered lung culture Length ofCondition Media Ventilation Perfusion culture Validation BGJb:Continuous; 2 ml/min 4-8 days DMEM + 10% FBS Once daily Perfusion DMEM +10% FBS None 2 ml/min 8 days Ventilation DMEM + 10% FBS Continuous 2ml/min 8 days with media Air-liquid DMEM + 10% FBS Continuous None 8days interface with media for 4 days, then air for 4 days Media DMEM +10% FBS Continuous None 8 days screen to BGJb transition

Demonstration of Epithelial Cell Repopulation

The results presented herein demonstrate the adherence and proliferationof epithelial cells on decellularized lung scaffolds. For theseexperiments, DMEM+10% FBS medium, perfusion of the vasculature at 2ml/min, once daily ventilation, uncoated decellularized scaffolds, andunsorted neonatal pulmonary cell populations (the ‘optimized’ conditionsdescribed elsewhere herein) was used.

FIG. 29 shows H&E staining of engineered lung tissue. At 4 days, asignificant number of organized, cuboidal-epithelial-lined developingepithelial structures was observed, while at 8 days, fewer suchstructures were observed and many cells adopted a more mature phenotype.At 4 days, many cells were proliferating, while at 8 days, fewerproliferating cells were observed (FIGS. 30A-B). At both 4 and 8 days,no significant numbers of apoptotic cells was observed (FIGS. 31A-B).

Immunofluorescence was used to document the expression of key epithelialcell markers, using aquaporin-5 for type I pneumocytes, surfactantprotein C for type II pneumocytes, and Clara cell secretory protein forClara cells. Type II epithelial cells generally predominated in thecultures, especially at later time points, as shown in FIGS. 33A-C.Clara cells were observed at high densities at 4 days, with fewer cellsat 8 days (FIGS. 32A-C). Staining for aquaporin at 4 days was alsoobserved, although significantly less aquaporin staining was observed atlater time points (FIGS. 34A-C).

It was observed that aquaporin staining for type I epithelium depictscells that are cuboidal in shape at 4 days of culture, which is contraryto their usual flat morphology, as the cells that line alveoli infunctioning lungs. In addition, these cuboidal cells also frequentlystained positive for either SPC or CCSP, as seen in FIGS. 36A-C and 37.Therefore, it is unlikely that the cells that express aquaporin at 4days are mature type I epithelium, as would be suggested by a flattenedmorphology and expression of aquaporin without other markers.

Type I pneumocytes are derived in vivo from type II epithelial cells,which are a locally resident precursor cell for alveolar epithelium. Innative lungs during development, type I pneumocytes do not achieve finaldifferentiation state in the absence of fetal breathing movements[Inanlou et al., 2005, Dev Dyn 233:772-82] and remain cuboidal in shapeon histology and TEM [Inanlou et al., 2005, Histol Histopathol20:1261-6].

The lack of type I pneumocyte differentiation was not surprising inthese cultures that were not regularly ventilated. The cells that doexpress aquaporin most likely arise from a local precursor cell that hadnot fully differentiated to type I epithelium. Therefore, thedecellularized scaffolds can support the attachment and proliferation ofpulmonary epithelium. Robust growth of type II epithelium, as well asClara cells and cells that are likely the precursors to fullydifferentiated type I epithelial cells was observed. We observe thesefindings under conditions of medium perfusion through the vasculaturewith only occasional breathing movements.

Epithelial Progenitor Cell Repopulation

Growth of two types of pulmonary epithelial progenitor cells on thedecellularized scaffolds was observed. Cells that are dual-positive forCCSP and SPC are reported to be local progenitor cells, termedbronchoalveolar stem cells, which can differentiate into both Claracells and type II pneumocytes and are found at the bronchoalveolar ductjunction [Lane et al., 2007, Regenerative Medicine 2:407-15; Kim et al.,2005, Cell 121:823-35]. FIGS. 35A-B shows such dual-positive cells,which are found in structures consistent with the appearance of terminalbronchioles, the expected physiological location of these cells.

Basal cells are a local stem cell for pulmonary airways; they residebelow the columnar epithelium and serve as a regenerative cell sourcefor epithelium of the proximal airways. FIGS. 36A-C demonstrates cellsthat are positive for cytokeratin-14, which is a basal cell marker. Forcomparison, native lung is shown in FIG. 36A. In addition, dual stainingfor cytokeratin-14 and CCSP is shown in FIG. 37 , which alsodemonstrates that the Clara cells are lining the airway and the basalcells lying beneath them, consistent with their normal anatomiclocations. These cells are sometimes found beneath larger airwaystructures, consistent with their location in native lung (FIG. 36B),but are also found in clusters that do not appear to be associated withlarge airways (FIG. 36C).

In addition to epithelial and endothelial cell growth, mesenchymal cellscan repopulate the decellularized lung scaffolds. FIGS. 38A-B showsimmunofluorescence staining of α-actin, which stains smooth muscle andmyofibroblasts. These engineered tissues were cultured under the sameconditions that were shown to favor epithelial growth. It was alsoobserved that mesenchymal cells were found to be located beneath andbetween the developing epithelial structures, consistent with theirlocation in native lung. Therefore, the results presented hereindemonstrate that the decellularized scaffolds are also suitablesubstrates for the growth of mesenchymal cells, and that the viablemesenchymal cells were contained within the population of neonatalpulmonary cells.

Effect of Media Composition on Epithelial Differentiation

Medium type can have significant effects on cell growth anddifferentiation, and thus on the development of engineered lung tissues.In order to investigate some of these differences in more detail, thegrowth of engineered lung cultures using a serum-free media (BGJb)versus a serum-containing media (DMEM+10% FBS) on epithelialdifferentiation was compared. In these experiments, cells were firstseeded onto the scaffolds in DMEM+10% FBS and allowed to culture in thismedium for 2 days, after which a gradual 4-day transition to BGJb(serum-free) media occurred, with the final 2 days of culture in pureBGJb media. The transition to serum-free medium caused substantialeffects on the expression of surfactant. It was observed that serum-freemedium lead to a more apical expression of surfactant (SPC) as comparedto DMEM+10% FBS (FIGS. 39C and 39D). This corresponds to significantlyincreased expression of surfactant on Western blot with the serum-freemedium (BGJb) (FIG. 40 ; compare lanes labeled ‘DMEM’ and ‘BGJb’). Inaddition, the form of surfactant was much more consistent with nativelung (with most surfactant noted as the 21 kDa pro-SPC form in BGJbmedium).

In addition, the transition to serum-free medium lead to a decrease inCCSP expression, noted via immunofluorescence in FIGS. 39F and 39E). Inboth medium types, diffuse CCSP expression was observed in the lumens ofthe developing epithelial structures. It is believed that this phenotypewas due to lack of perfusion or ventilation, as these cultures wereperformed in small tissue slices.

Effect of Air-Liquid Interface on Lung Development in the Bioreactor

In order to create an air-liquid interface, the engineered lungs werecultured first for 4 days under ventilation with media, to allow cellattachment and proliferation. For the final 4 days of culture, theventilation was switched from media to filtered room air.

Ventilation with air caused severe damage to the airway epithelium aswell as destructive changes to some alveolar walls (FIGS. 19A-C).Therefore, the tidal volume used for air ventilation was reduced byapproximately 50%, from the previous value of ˜2.0-2.5 ml for liquid orair breathing in an effort to reduce the damage caused to native lungsby air ventilation.

Changes in cell attachment or morphology due to the presence of airventilation in the bioreactor was not observed. However, it was observedthat ventilation with air in engineered lungs cultured in the bioreactorled to induced expression of aquaporin, a differentiation marker fortype I epithelium. This was noted both in cells in the parenchyma, whichtypically stained solely for surfactant protein C (indicate of type IIepithelium) as well as occasional staining of cuboidal cells indeveloping epithelial structures. The observed staining patternsindicate that it is highly likely that most of the aquaporin-expressingcells also express SPC. As can be seen from FIG. 42B, virtually all ofthe cells in the parenchyma express SPC, while a subset of theparenchymal cells express AQP (FIG. 41A).

These findings are highly suggestive that the air-liquid interface isinducing differentiation of type II epithelium to type I epithelium.Type II epithelium is a known local progenitor cell for type Iepithelium, and so this differentiation is not surprising [Adamson etal., 1974, Lab Invest 30:35-42; Fehrenbach, 2001, Respir Res 2:33-46].Furthermore, reduced expression of surfactant protein C inair-ventilated engineered lungs was observed, as shown in FIG. 40(compare lanes ‘ALI’ to lungs that were ventilated with medium (Vent′)and perfused (Ted)). This is also consistent with the differentiation oftype II to type I epithelium. In addition, the growth of a number ofciliated epithelial cells was observed, as shown in FIGS. 42A-B. Thislikely a result of the introduction of an air interface. When airwayepithelium is cultured in vitro, the transition of the cells from liquidto the air interface induces cilia expression [You et al., 2002, Am JPhysiol Lung Cell Mol Physiol 283:L1315-21; Davidson et al., 2004, JCyst Fibros 3 Suppl 2:59-62]. Furthermore, the lack of an air-liquidinterface can lead to reduced ciliogenesis [Ostrowski et al., 1995, ExpLung Res 21:957-70; Yeh et al., 2007, Laryngoscope 117:1439-44].Therefore, in some instances, air interface has important impacts onregeneration of lung tissue in vitro.

Effect of Perfusion and Ventilation on the Development of EpithelialStructures in Engineered Lung Tissues

Perfusion and ventilation can have significant impacts on the culture oflung tissue in the bioreactor. Ventilation also has significant impactson the lung epithelial development, including the differentiation oftypes I and II pneumocytes [Inanlou et al., 2005, Histol Histopathol20:1261-6; Inanlou et al., 2005, Dev Dyn 233:772-82; Inanlou et al.,2005, Dev Dyn 232:43-54]. As a result, the effects of perfusion andventilation on engineered lung development during 8-day cultures in thebioreactor were compared. For these experiments, the conditions were thesame as utilized during the validation experiments discussed elsewhere,with culture medium of DMEM+10% FBS, uncoated scaffolds, and an unsortedneonatal pulmonary cell population. However, cultures were eitherperfused through the vasculature at 2 ml/min or ventilated continuouslywith medium at 1 breath/min.

Many of the cells that form epithelial structures stain positive forCCSP, as shown in FIG. 50 . Staining for CCSP in ventilated cultureswere observed, but the cells attained a more flattened morphology. Thismay indicate that ventilation is inducing the CCSP-expressing cells todifferentiate towards alveolar epithelium (either type I or IIpneumocytes).

In the absence of ventilation, the lumens of developing epithelialstructures were filled with an eosinophilic, and thus likelyproteinaceous, material, visible on H&E staining in FIGS. 43A-B. Thismaterial also stained positive for CCSP, as seen in FIGS. 45A-B. Thisbuildup of CCSP indicates that the Clara cells lining these epithelialstructures are producing CCSP. In addition, the removal of this materialwith ventilation indicates that the airway tree is still intact and canconduct fluid, and furthermore suggests that these developing epithelialstructures are a part of the airway tree and do not proliferate randomlywithin the matrix.

Perfusion versus ventilation did not have significant effects on theexpression of SPC, as shown in FIGS. 46A-B. The majority of cells werepositive for SPC under both ventilation and perfusion conditions.

Example 5: Endothelial Development in Engineered Lung Tissues

The following experiments were designed to determine whetherdecellularized scaffolds are able to support the growth of engineeredlung endothelium, and to evaluate the effects of several specificfactors on the development of engineered endothelium, with a focus onthe ability of these factors to impact the formation of a functionalendothelial barrier between the vascular and airway compartments.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

Scaffold Preparation

Decellularized scaffolds were prepared as described elsewhere herein.

Neonatal Cell Isolation and Seeding

Neonatal rat pulmonary cells were isolated as described elsewhereherein. Cells were seeded into the scaffolds as described elsewhereherein.

Endothelial Cell Culture

Rat lung microvascular endothelial cells were obtained from VECTechnologies (Renssalaer, N.Y.) and grown on fibronectin-coated (˜1μg/cm2, Gibco) tissue culture vessels in MCDB-131 complete mediaincluding 10% FBS and supplemental growth factors (VEC Technologies).

Optimized Conditions for Engineered Endothelial Culture

Scaffolds were coated with 1 mg of fibronectin (Gibco) perfused throughthe vasculature in 60 ml of PBS at 37° C., then rinsed with PBS andmedia. Each scaffold was seeded twice at days 0 and 2 or 3 of culturewith 8-10 million rat lung microvascular EC at each time point (two T150culture flask was used per lung for each of two seedings). Cells weretrypsinized from tissue culture plates using 0.25% tryspin (Gibco),filtered through a 40 μm filter to remove cell clumps, and injected intothe pulmonary artery as a single bolus injection in ˜3 ml of media.After allowing cell adherence for 1 hour, perfusion was begun throughthe vasculature at ˜1.5 ml/min. After 1-2 hours, the perfusion rate wasincreased to 3 ml/min for the remainder of the culture period of 7-10days. Medium was changed every 3-4 days.

Immunofluorescence

Tissue samples were prepared and stained as described elsewhere herein.

Transmission Electron Microscopy (TEM)

Samples were prepared and analyzed as described elsewhere herein.

Microparticle Retention

An assay was developed to evaluate the permeability of whole rat lungsto smaller particles, which have sizes on the order of largemacromolecules. In this assay, the leakage of a FITC-labelled dextransolution across the airway-vascular barrier was quantified.FITC-labelled dextran with a molecular weight of 2,000,000 Da wasobtained from Sigma (St Louis, Mo.). Assay validation was performed bymeasuring the permeability of native lung and native lung that wastreated with 0.025% trypsin for 2 min. Lungs were perfused withheparinized PBS and connected to the usual bioreactor cannulae. Abaseline lavage sample was obtained, and then the trypsin-treated lungwas perfused with 10 ml of 0.025% trypsin in PBS and allowed to dwellfor 2 min at RT, then rinsed with 10 ml of PBS. The FITC-labelleddextran solution (1 mg/ml) was injected into the pulmonary artery, andthen flushed with 20 ml of PBS. Two lavage samples were then immediatelytaken in succession from the trachea. Fluorescence was measured using afluorescent plate reader and data were fit to a standard curve. Whenperformed on decellularized or engineered lungs, the assay was performedthrough the airway, as with the microsphere assay (see section 3.2.9).Thus, the FITC-dextrans were injected into the airway, and thevasculature was flushed with PBS.

The results of the experiments are now described.

Cell source: A commercially purchased source of rat lung microvascularEC was used. When these cells were seeded into the scaffolds that werefibronectin-coated and cultured in the presence of EC-specific medium,substantial growth of endothelial cells was observed, as shown in FIG.47 .

Summary of endothelial screening experiments: The screening experimentsdiscussed elsewhere herein allowed for the identification of a set ofconditions that was compatible with engineered endothelial culture.Outcomes were assessed primarily via histology for cell viability andexpression of PECAM on immunofluorescence. The result of these pilotstudies was a set of conditions that enables endothelial cell growthinside the scaffolds, such that the impact of discrete conditions onengineered lung endothelium can be systematically evaluated.

The conditions that were identified as suitable for the culture ofengineered endothelium were: the use of a purified, in vitro expandedpopulation of rat lung microvascular EC; fibronectin-coated scaffolds;and the use of EC-specific medium (MCDB-131 with 10% FBS andsupplemental growth factors).

Validation of FITC-Dextran Permeability Assay

An assay to evaluate the permeability of whole rat lungs to smallparticles, which have sizes on the order of large macromolecules wasdeveloped. In this assay, the leakage of a FITC-labelled dextransolution across the airway-vascular barrier was quantified. This assaycan be used repeatedly over the course of a culture, involves materialsthat are cell culture-friendly, and provides a measure of thepermeability of the entire lung. In addition, if the assay is performedimmediately before fixation, the FITC-dextran could be identified onhistologic sections using anti-FITC antibodies.

The FITC-labelled dextran has a molecular weight of 2,000,000 Da. For amono-disperse dextran, the Stokes-Einstein radius (nm) is related tomolecular weight by rs=0.0488(MW)0.437 [Venturoli et al., 2005, Am JPhysiol Renal Physiol 288:F605-13; Oliver et al., 1992, J Am Soc Nephrol3:214-28]. For a 2 MDa dextran, this yields a radius of 27.7 nm. Thisassay was validated by evaluating the permeability of native lung andnative lung that was made ‘leaky’ by brief perfusion of the vasculaturewith dilute trypsin. The FITC-labelled dextran solution was injectedinto the pulmonary artery, and then flushed with saline. Two lavagesamples were then immediately taken in succession from the trachea. Asshown in FIGS. 48A-B, the permeability of lung is increased by trypsintreatment, as expected due to disruption of endothelial attachment tothe basement membrane. However, even native lung provided a measurableleak via this assay. This degradation of vascular permeability is theresult of delays between animal sacrifice and the injection of thedextran, as well as the handling of the lung tissue.

When performed on decellularized or engineered lungs, the assay wasperformed through the airway, as with the microsphere assay. Thus, theFITC-dextrans were injected into the airway, and the vasculature wasflushed with saline. Dextrans that translocated into the vascularcompartment were measured as leak. The assays were performed in thisfashion because in decellularized or engineered lungs, the tissue ishighly permeable to fluid and a return sample cannot be obtained afteran airway lavage. As such, the dextran was injected into the air-way asa single bolus lavage, and the vasculature was flushed to measure leakof FITC-dextrans across the airway-vascular barrier.

Effects of Perfusion Versus Ventilation on Engineered Lung Endothelium

The following experiments where designed to evaluating the effects ofspecific conditions on the development of engineered endothelialtissues, with a focus on the formation of a functional endothelialbarrier. The effects of culturing engineered lung endothelium withperfusion versus ventilation was compared. Both ventilation andperfusion with regards to endothelial cell survival and proliferationand the formation of cell-cell junctions using transmission EM wasevaluated.

It was observed that perfusion substantially improved the growth ofengineered lung endothelium, as shown on histology in FIGS. 48A-B. Inaddition, more apoptotic cells were observed with ventilation, as shownin FIGS. 49A-B and consistent with their poor appearance on H&Ehistology.

In addition, perfused and ventilated cultures were analyzed for thepresence of cell junctions using transmission EM and VE-cadherinstaining. Tight junctions between endothelial cells are an importantmeans of barrier function, as they tightly link adjoining cells togetherand thus inhibit the movement of fluid between these paracellular spaces[Majno et al., 1961, J Biophys Biochem Cytol. 11:571-605]. If these celljunctions are weak or absent, fluid leak can occur out of thevasculature and cause pulmonary edema [Orfanos et al., 2004, IntensiveCare Med 30:1702-14; Maniatis et al., 2008, Vascular Pharmacology49:119-33].

Using TEM, cell-cell junctions in the perfused engineered tissues wereobserved, as shown in FIG. 50 . Not all cells demonstrated tightjunction formation.

Cell junction formation was assessed using immunofluorescence forVE-cadherin. Robust staining for VE-cadherin was found in perfusedengineered lung endothelium, as shown in FIGS. 51A-B. Ventilated tissueswere not stained for VE-cadherin due to their poor appearance onhistology and TEM.

Assessment of Barrier Function of Engineered Lung Endothelium

The following experiments were designed to evaluate the ability ofengineered lung endothelium to form a functional barrier between thevascular bed and the airspaces. This is important in order to reducefluid leak into the alveoli and thus enable gas exchange, and it is akey component of the objectives for engineered lung tissue. In order toevaluate the barrier function provided by the lung endothelium, apermeability assay was used to measure the translocation of small (55nm) FITC-dextran particles from the airspaces into the vasculature. Thisassay was developed and validated as described elsewhere herein.

For this assay, FITC-dextran was injected into the airway compartment,and the amount that leaked across the alveolar-vascular barrier wasmeasured by flushing the vasculature with saline. For decellularizedscaffolds, there was essentially no barrier function to such smallparticles, with virtually all (98.4%) of the dextran translocating thealveoli into the vasculature and recovered with vascular rinsing. Thiscompares to native lung, which when treated similarly shows a leak of12.9% (FIG. 52 ).

In engineered lung endothelial tissues that were perfused, retention ofup to 30% of dextrans in the airway compartment was demonstrated, afterculture periods of 7-10 days. Ventilated endothelial tissuesdemonstrated a permeability of 87%.

These findings for perfused engineered lung endothelium, especially whencoupled with the findings of robust VE-cadherin expression viaimmunofluorescence (FIGS. 51A-B) and cell-cell junction formation viaTEM, indicate that the formation of a functional endothelial barrier inengineered endothelial tissues has occurred.

FIG. 63 is a chart depicting the ultimate tensile strength of engineeredtissues. Native and decellularized lung strengths are also shown. Thisfigure shows that the ultimate tensile stress of the engineered tissuesis comparable to both native lung and to decellularized matrix.Therefore, the mechanical properties of tissue engineered lungs aremaintained after the recellularization process.

Example 6: Lung Cell Therapy

The results presented herein demonstrate the use of decellularized lungtissues to effect lung cell therapy in a mammal. Generally, the stepsinclude decellularization of a trachea, detection of extracellularmatrix components within the decellularized tracheal tissue, culture ofeither human bronchial (large airway) and small airway pulmonaryepithelial cells on the decellularized tracheal matrix, gene therapy ofhuman pulmonary epithelial cells with a desired gene, and instillationof human pulmonary epithelial cells into lung via instillation, withverification of cell attachment and survival in the recipient lung.

Decellularization of Trachea

Pig trachea was harvested and rinsed in PBS to remove blood. A piece wascut and fixed with 10% neutral-buffered formalin, embedded in paraffinand cut into 5-mm sections. The rest was cut into 5 rings and incubatedin CHAPS buffer (pH13.5) with stirring for 2-24 hours, with CHAPS bufferchanged at 2 h, 4 h and 8 h time points. At the indicted time, tissuewas removed from CHAPS buffer and rinsed with PBS. A piece was cut andfixed with 10% formalin and processed for histological analysis toconfirm decellularization.

It was observed that decellularized trachea prepared with incubation inCHAPS buffer for 4-8 hours maintained collagen matrix and had most ofcells removed from the tissue (except the cartilage layer). See FIG. 55. The following experiments were based on 6 h CHAPS incubation withstirring for preparation of decellularized trachea.

These experiments were designed to detect extracellular matrix intrachea before and after decellularization. Briefly, a piece of tissuewas cut from native and decellularized trachea and fixed with 10%formalin. 5 μm paraffin-embedded sections were stained with H&E andMasson's Trichrome. Paraffin-embedded sections were also immunostainedfor extracellular matrix proteins including collagen (COL), fibronectin(FN) and laminin (LN). Sections were deparaffinized, rehydrated, antigenretrieved (using Proteinase K) and blocked. In-house-raised rabbitanti-human FN, COL I, COL III, COL IV, COL V or LN antibodies wereapplied to sections, followed by Alexa Fluor 546-conjugated goatanti-rabbit IgG. Slides were counterstained with DAPI. It was observedthat native trachea stained positive for COL I, COL III, COL V, but notthe other ECM proteins. Decellularized trachea contained all the threetypes of COL seen in native trachea. See FIG. 56 . These results suggestthat decellularized trachea tissue support airway epithelial celladhesion, growth and differentiation in vitro.

Growing Airway Epithelial Cells on Decellularized Trachea Tissue

The next set of experiments was designed to grow cells on decellularizedtissue. Briefly, human bronchial/tracheal epithelial cells (NHBE) werecultured in bronchial epithelial growth medium (BEGM). Decellularizedtrachea (6 h CHAPS buffer incubation) was rinsed extensively (at least24 h) with sterile PBS. The cartilage layer (as well as the adventitiallayer) was peeled off, leaving the trachea mucosal and submucosal layerfor subsequent cell seeding. Tissue was cut to about 5×5 cm² in size andput on transwell insert (0.4 μm pore size) in 6-well plate, with theepithelial surface facing up.

Cells were seeded on decellularized trachea tissue as follows. 50 μlcells (at 3.5×10⁶ cells/ml) were added to the epithelial surface of thedecellularized trachea tissue and incubated for 30 mins at 37° C. Freshculture media was then added to both the upper and lower sides oftranswell insert, and cells were incubated for additional time. At theindicated time, tissues were removed from the transwell and fixed with10% formalin. Paraffin-embedded 5 μm sections were stained with H&E. Itwas observed that decellularized trachea supported NHBE adhesion andgrowth. See FIG. 57 .

The next set of experiments were designed to grow human small airwayepithelial cells (SAEC) that were transfected with a transgene ondecellularized tissue. Briefly, human SAECs were transfected with GFPRetrovirus for 6 h. Cells were seeded on decellularized trachea tissuein 6-well plates as follows. 50 μl cells (at 5×10⁶ cells/ml) were addedto the epithelial surface of the decellularized trachea tissue (about0.5×1.0 cm²) and incubated for 30 min at 37° C. Fresh culture media werethen added and cells were incubated for additional time. It was observedthat decellularized trachea also supported SAEC adhesion and growth. SeeFIG. 58 . In addition, decellularized trachea supported the adhesion andgrowth of SAEC that had been transfected with GFP, as aproof-of-principle of culturing human pulmonary epithelial cells thathave been transfected with a gene of interest.

Gene Therapy of Airway Epithelial Cells

The next set of experiments was designed to determine the feasibility ofusing the decellularized tissue for gene therapy for lung cells. Geneticmodification was performed as follows. EGFP (enhanced GFP) retrovirussupernatant was prepared using Phoenix packaging cell line. EGFP DNA wasinserted in the LZRSpBMN vector. NHBE and SAEC were grown until over 80%confluent. On the day of infection, cells were rinsed a few times andinoculated with virus supernatant (containing 8 μg/ml polybrene) for 6 hat 37° C. Cells were rinsed a few times and incubated in fresh mediaovernight. Cells were then analyzed for GFP using flow cytometry. It wasobserved that the infected cells stained positive for GFP. For example,NHBE cells exhibited an 18.5% positive staining for GFP compared tonon-infected cells. SAEC cells exhibited a 16% positive staining for GFPcompared to non-infected cells. The results presented herein demonstratethe ability to infect human pulmonary epithelial cells with a gene ofinterest in this culture system, using a retrovirus. However, othermeans of delivering a transgene is also encompassed in the invention.For example, the next set of experiments were designed to test thefeasibility of using a lentivirus system.

Infection with GFP lentivirus was performed as follows. EGFP Lentivirussupernatant was prepared in 293T cells in serum free medium. GFP DNA wasinserted in the pSicoR vector with CMV promoter. NHBE were seeded onto6-well plate at 1×10⁵ cells/well and incubated at 37° C. for 1 day(80-90% confluent). On day of infection, cells were rinsed a few timesand then inoculated with virus supernatant (1:3 diluted with fresh BEGM)(containing 8 μg/ml polybrene) for 6 h at 37° C. Cells were rinsed a fewtimes and then incubated with fresh media for additional 2 days. Cellswere fixed with 4% paraformaldehyde for 30 min and examinedmicroscopically. See FIG. 59 . The results are summarized in Table 2:

TABLE 2 Total GFP + Infection Sample cells cells % Control 0.996 × 10⁶ 0 6 h 1.190 × 10⁶ 0.635 × 10⁶ 52.7 20 h 0.972 × 10⁶ 0.638 × 10⁶ 64.9 6h + 0.727 × 10⁶ 0.389 × 10⁶ 53.6 6 h 20 h + 0.420 × 10⁶ 4564 10.7 20 h

The results presented herein demonstrate that NHBE infected with GFPlentivirus did not show obvious morphology change after 6 h. One-timeinfection for 6 h produced over 50% GFP positive cells assessed by flowcytometry. Infection efficiency for SAEC also appeared to be over 50%.The results presented herein demonstrate that any desired gene can beused to generate genetically modified lung cells.

Injection of Airway Epithelial Cells into Mouse Lungs

The next experiments were designed to determine the feasibility ofinjecting airway epithelial cells into a mammalian lung. Briefly, 100 μlof 1:1 mixture of 5 μm microspheres and PBS was injected through tracheainto C57BL/6J mouse, female, ˜10 weeks old (Jackson Lab). The mousesurvived for a few minutes after injection. Lung was harvestedimmediately and fixed with formalin. 5 μm paraffin-embedded sectionswere stained with H&E. This study was done as an initial feasibilitystudy to determine if cell-sized particles that were delivered byinstillation into the airway would be detected within mammalian lungs.Results from H&E images of mouse lung with injected microspheresdemonstrated that significant numbers of microspheres were present inevery lobe of the mouse lung. See FIG. 60 . The results demonstrate thatinjection using the trachea approach works for cell instillation.

The next set of experiments included injecting GFP labeled cells intoexplanted mouse lungs. Briefly, cells were infected with GFP retroviruswith 6 h. C57BL/6J mice were euthanized, trachea exposed and theproximal end tied off with suture. 100 μl of GFP retrovirus-infectedSAEC were injected through trachea (followed by 200 μl air to push thecells in) into the lungs of mice. The distal end of the trachea was tiedoff. Lung was explanted and fixed immediately with 10% formalin. 5 μmparaffin-embedded sections were deparaffinized, rehydrated, antigenretrieved (using 10 mM Citric Acid buffer, pH 6), permeabilized (withTriton X) and blocked. Rabbit polyclonal anti-GFP antibody (from Abcam)was applied to sections, followed by Alexa Fluor 555-conjugated goatanti-rabbit IgG. Slides were counterstained with DAPI. It was observedthat GFP positive cells were found in the explanted lungs. See FIG. 61 .The results presented herein demonstrate the successful injection ofcells into the lungs, and that human epithelial cells that have beentransduced with a transgene (GFP) adhered to lung epithelium.

The next set of experiments was designed to determine the feasibility ofinjecting of GFP-labeled cells into mouse lungs. Briefly, mice(C57BL/6J, female, ˜10 weeks old) were anesthetized and trachea wasexposed. 100 μl of GFP retrovirus infected-human airway epithelial cellswere injected through trachea (followed by 200 μl air) into the lungs.See FIG. 62 . Mice were allowed to recover for 6 h to 3 days. On day ofharvest, the lung was perfused with PBS through pulmonary artery toremove blood, and then perfused with 10% formalin through trachea, anddissected. Harvested lung were fixed in formalin for another 4 hr. 5 μmparaffin-embedded sections were stained for GFP as discussed elsewhereherein. It was observed that GFP positive human airway epithelial cells(both NHBE and SAEC) were found in mouse lungs for days afterinstillation into the airway. See FIG. 63 . This shows that humanepithelial cells, that have been grown in culture and have beentransduced with a transgene of interest, can be delivered into the lungsof a recipient mammal, and adhere to the recipient lung and survive andproliferate.

Example 7: Implantation of Decellularized Engineered Lung

This experiment was designed to show the feasibility of implanting adecellularized, engineered lung into a living rat recipient. Adecellularized engineered rat lung was prepared according to previousexamples. An adult male laboratory rat was anesthetized withintraperitoneal injection of ketamine and xylazine. The rat was thentracheally intubated and ventilated with 100% oxygen mixed with Foraneto maintain anesthesia. Under sterile conditions, the thorax was openedvia median sternotomy. The ribs were retracted bilaterally, revealingnormally inflating lungs and the beating heart. Following systemicheparinization, the native left lung was excised in toto. Then, thedecellularized engineered lung was anastomosed to the recipient'spulmonary artery, pulmonary vein, and left mainstem bronchus using 10-0suture under an operating microscope.

After removal of vascular clamps on the pulmonary artery and vein, bloodwas seen to perfuse the engineered lung in normal fashion. In addition,the implanted engineered lung was easily ventilated and cycled throughinflation and deflation similar to the resident native right lung. FIGS.64A-B shows photographs of the implanted engineered lung at inflationand deflation during the ventilatory cycle. Hence, the engineered lungsproduced using the techniques herein are both implantable into a mammal,and are functional from the standpoint of enabling perfusion through thewhole organ, and easy ventilation of the airway. During the entireimplantation period, there was no evidence of bleeding or air leak fromthe implanted engineered lung.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1-39. (canceled)
 40. A method for making an engineered three dimensionaltissue capable of supporting and maintaining the differentiation stateof a lung cell, said method comprising seeding a decellularized scaffoldwith a population of cells to produce a seeded scaffold.
 41. The methodof claim 40, wherein said decellularized scaffold exhibits an intactairway tree and vascular network.
 42. The method of claim 40, whereinsaid population comprises a stem cell.
 43. The method of claim 40,wherein said population comprises epithelial and endothelial cells. 44.The method of claim 40, wherein said cells are genetically modified. 45.The method of claim 40, wherein said decellularized scaffold is capableof supporting and maintaining the differentiation state of an alveolarepithelial cell.
 46. The method of claim 40, wherein said scaffoldcomprises a biocompatible material selected from the group consisting offibronectin, laminin, collagen, glycoprotein, thrombospondin, elastin,fibrillin, mucopolysaccharide, glycolipid, heparin sulfate, chondroitinsulfate, keratin sulfate, glycosaminoglycan, hyaluronic acid,proteoglycan, vitronectin, poly-D-lysine, polysaccharide, andcombinations thereof.
 47. The method of claim 40, comprising cells thatexhibit gene expression associated with induction of branchingmorphogenesis.
 48. The method of claim 40, wherein said gene is cysticfibrosis transmembrane conductance regulator (CFTR).
 49. The method ofclaim 40, wherein said engineered three dimensional tissue exhibits acharacteristic of a lung tissue, wherein said characteristic is selectedfrom the group consisting of branching morphogenesis, distal lungepithelial cytodifferentiation, epithelial growth, vascular development,and combinations thereof.
 50. An in vitro method for screening a testagent for the ability of said test agent to modulate the health of alung tissue, said method comprising contacting said test agent to anengineered three dimensional lung tissue model and measuring the effectsaid test agent has on said model, wherein any alteration to the modelis an indication that said test agent is able to modulate the health ofa lung tissue.
 51. The method of claim 50, wherein said engineered threedimensional tissue is derived from a decellularized scaffold.
 52. Themethod of claim 50, wherein the test agent is selected from the groupconsisting of a chemical agent, a pharmaceutical, a peptide, a nucleicacid, and radiation.
 53. The method of claim 50, wherein the test agentis a delivery vehicle for a therapeutic agent.
 54. The method of claim50 comprising determining the effect of the test agent on cell number,area, volume, shape, morphology, marker expression or chromosomalfragmentation.
 55. The method of claim 50, further comprising the stepof selecting an agent which has a desired effect on the lung tissuemodel. 56-58. (canceled)
 59. An implantable composition comprising adecellularized tissue capable of supporting cell growth, wherein thedecellularized tissue exhibits a characteristic of a correspondingnatural tissue prior to decellularization.